Replication competent attenuated vaccinia viruses with deletion of thymidine kinase with and without the expression of human FLT3L or GM-CSF for cancer immunotherapy

ABSTRACT

The present invention relates generally to the fields of oncology, virology and immunotherapy. More particularly, it concerns the use of poxviruses, specifically the replication competent attenuated vaccinia virus with deletion of thymidine kinase (VC-TK − ) with and without the expression of human Flt3L or GM-CSF as oncolytic and immunotherapy. The foregoing poxviruses can also be used in combination with immune checkpoint blocking agents. The foregoing poxviruses can also be inactivated via Heat or UV-treatment and the inactivated virus can be used as immunotherapy either alone or in combination with immune checkpoint blocking agents.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/079,217, filed Aug. 23, 2018, which is a National Stage Applicationof PCT/US2017/019548, filed Feb. 25, 2017, which claims the benefit ofand priority to U.S. Provisional Application Ser. No. 62/300,066, filedFeb. 25, 2016, the entire contents of each of which are incorporatedherein by reference.

GOVERNMENT SUPPORT

The invention was made in part with government support under grantsAI073736, AI095692, CA008748 and CA56821 awarded by the NationalInstitutes of Health. The U.S. government has rights in this invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 23, 2017, isnamed 11000_005154-WO0_SL.txt and is 2,658 bytes in size.

FIELD OF THE INVENTION

The present invention relates generally to the fields of oncology,virology and immunotherapy. More particularly, it concerns the use ofpoxviruses, specifically the replication competent but attenuatedvaccinia virus (1000 times attenuated compared to wild type vaccinia andtherefore safe) with deletion of thymidine kinase (VC-TK⁻) with andwithout the expression of human Flt3L or GM-CSF as oncolytic andimmunotherapy. (VC-TK⁻ is 1000 times less virulent compared to wild typevaccinia and therefore safe) The foregoing poxviruses can also be usedin combination with immune checkpoint blocking agents. The foregoingpoxviruses can also be inactivated via Heat or UV-treatment and theinactivated virus can be used as immunotherapy either alone or incombination with immune checkpoint blocking agents.

BACKGROUND

Immune System and Cancer

Malignant tumors are inherently resistant to conventional therapies andpresent significant therapeutic challenges. Immunotherapy has become anevolving area of research and an additional option for the treatment ofcertain types of cancers. The immunotherapy approach rests on therationale that the immune system may be stimulated to identify tumorcells, and target them for destruction.

Numerous studies support the importance of the differential presence ofimmune system components in cancer progression (Jochems and Schlom, ExpBiol Med, 236(5): 567-579 (2011)). Clinical data suggest that highdensities of tumor-infiltrating lymphocytes are linked to improvedclinical outcome (Mlecnik et al., Cancer Metastasis Rev.; 30: 5-12,(2011)). The correlation between a robust lymphocyte infiltration andpatient survival has been reported in various types of cancer, includingmelanoma, ovarian, head and neck, breast, urothelial, colorectal, lung,hepatocellular, gallbladder, and esophageal cancer (Angell and Galon,Current Opinion in Immunology, 25:1-7, (2013)). Tumor immune infiltratesinclude macrophages, dendritic cells (DC), mast cells, natural killer(NK) cells, naive and memory lymphocytes, B cells and effector T cells(T lymphocytes), primarily responsible for the recognition of antigensexpressed by tumor cells and subsequent destruction of the tumor cellsby T cells.

Despite presentation of antigens by cancer cells and the presence ofimmune cells that could potentially react against tumor cells, in manycases the immune system does not get activated or is affirmativelysuppressed. Key to this phenomenon is the ability of tumors to protectthemselves from immune response by coercing cells of the immune systemto inhibit other cells of the immune system. Tumors develop a number ofimmunomodulatory mechanisms to evade antitumor immune responses. Forexample, tumor cells secrete immune inhibitory cytokines (such as TGF-β)or induce immune cells, such as CD4⁺ T regulatory cells and macrophages,in tumor lesions to secrete these cytokines. Tumors have also theability to bias CD4⁺ T cells to express the regulatory phenotype. Theoverall result is impaired T-cell responses and induction of apoptosisor reduced anti-tumor immune capacity of CD8⁺ cytotoxic T cells.Additionally, tumor-associated altered expression of MEW class I on thesurface of tumor cells makes them ‘invisible’ to the immune response(Garrido et al. Cancer Immunol. Immunother. 59(10), 1601-1606 (2010).Inhibition of antigen-presenting functions and dendritic cell (DC)additionally contributes to the evasion of anti-tumor immunity (Gerliniet al. Am. J. Pathol. 165(6), 1853-1863 (2004).

Moreover, the local immunosuppressive nature of the tumormicroenvironment, along with immune editing, can lead to the escape ofcancer cell subpopulations that do not express the target antigens.Thus, finding an approach that would promote the preservation and/orrestoration of anti-tumor activities of the immune system would be ofconsiderable therapeutic benefit.

Immune checkpoints have been implicated in the tumor-mediateddownregulation of anti-tumor immunity. It has been demonstrated that Tcell dysfunction occurs concurrently with an induced expression of theinhibitory receptors, CTLA-4 and programmed death 1 polypeptide (PD-1),members of the CD28 family receptors. Nevertheless, despite extensiveresearch in recent years, the success of immunotherapy in a clinicalsetting has been limited. Few therapeutic agents have been approved byregulatory authorities, and among those, the benefit has been observedonly in a minority of patients. In recent years, immune checkpoints havebeen implicated in the downregulation of anti-tumor immunity and used astherapeutic targets. Studies have shown that T cell dysfunction occursconcurrently with an induced expression of the inhibitory receptor,programmed death 1 polypeptide (PD-1). PD-1 is an inhibitory member ofthe CD28 family of receptors that in addition to PD-1 includes withoutlimitation CD28, CTLA-4, ICOS and BTLA. However, to date, theseapproaches have met with limited success. While promise regarding theuse of immunotherapy in the treatment of melanoma has been underscoredby the clinical use and even regulatory approval of anti-CTLA-4(ipilimumab) and anti-PD-1 drugs (pembrolizumab and nivolumab) theresponse of patients to these immunotherapies has been limited. Recentclinical trials, focused on blocking these inhibitory signals in T cells(e.g., CTLA-4, PD-1, and the ligand of PD-1 PD-L1), have shown thatreversing T cell suppression is critical for successful immunotherapy(Sharma and Allison, Science 348(6230), 56-61 (2015); Topalian et al.,Curr Opin Immunol. 24(2), 202-217 (2012)). These observations highlightthe need for development of novel therapeutic approaches for harnessingthe immune system against cancer.

Melanoma

Melanoma, one of the most deadly cancers, is the fastest growing cancerin the US and worldwide. Its incidence has increased by 50% among youngCaucasian women since 1980, primarily due to excess sun exposure and theuse of tanning beds. According to the American Cancer Society,approximately 76,380 people in the US will be diagnosed with melanomaand 10,130 people (or one person per hour) are expected to die ofmelanoma in 2016. In most cases, advanced melanoma is resistant toconventional therapies, including chemotherapy and radiation. As aresult, people with metastatic melanoma have a very poor prognosis, witha life expectancy of only 6 to 10 months. The discovery that about 50%of melanomas have mutations in BRAF (a key tumor-promoting gene) openedthe door for targeted therapy in this disease. Early clinical trialswith BRAF inhibitors showed remarkable, but unfortunately notsustainable, responses in patients with melanomas with BRAF mutations.Therefore, alternative treatment strategies for these patients, as wellas others with melanoma without BRAF mutations, are urgently needed.

Human pathological data indicate that the presence of T-cell infiltrateswithin melanoma lesions correlates positively with longer patientsurvival (Oble et al. Cancer Immun. 9, 3 (2009)). The importance of theimmune system in protection against melanoma is further supported bypartial success of immunotherapies, such as the immune activatorsIFN-α2b and IL-2 (Lacy et al. Expert Rev Dermatol 7(1):51-68 (2012)) aswell as the unprecedented clinical responses of patients with metastaticmelanoma to immune checkpoint therapy, including anti-CTLA-4 andanti-PD-1/PD-L1 either agent alone or in combination therapy (Sharma andAllison, Science 348(6230), 56-61 (2015); Hodi et al., NEJM 363(8),711-723 (2010); Wolchok et al., Lancet Oncol. 11(6), 155-164 (2010);Topalian et al., NEJM 366(26), 2443-2454 (2012); Wolchok et al., NEJM369(2), 122-133 (2013); Hamid et al., NEJM 369(2), 134-144 (2013); Tumehet al., Nature 515(7528), 568-571 (2014). However, many patients fail torespond to immune checkpoint blockade therapy alone. The addition ofvirotherapy might overcome resistance to immune checkpoint blockingagents, which is supported by animal tumor models (Zamarin et al., SciTransl Med 6(226), 2014).

Poxviruses

Poxviruses, such as engineered vaccinia viruses, are in the forefront asoncolytic therapy for metastatic cancers (Kim et al., 2009). Vacciniaviruses are large DNA viruses, which have a rapid life cycle (Moss etal., 2007). Poxviruses are well suited as vectors to express multipletransgenes in cancer cells and thus to enhance therapeutic efficacy(Breitbach et al., 2012). Preclinical studies and clinical trials havedemonstrated efficacy of using oncolytic vaccinia viruses and otherpoxviruses for treatment of advanced cancers refractory to conventionaltherapy (Park et al., 2008; Kim et al., 2007; Thorne et al., 2007).Poxvirus-based oncolytic therapy has the advantage of killing cancercells through the combination of cell lysis, apoptosis, and necrosis. Italso triggers innate immune sensing pathway that facilitates therecruitment of immune cells to the tumors and the development ofanti-tumor adaptive immune responses. The current oncolytic vacciniastrains in clinical trials (JX-594, for example) use wild-type vacciniawith deletion of thymidine kinase to enhance tumor selectivity, and withexpression of transgenes such as granulocyte macrophage colonystimulating factor (GM-CSF) to stimulate immune responses (Breitbach etal., 2012, Curr Pharm Biotechnol). Many studies have shown however thatwild-type vaccinia has immune suppressive effects on antigen presentingcells (APCs) (Engelmayer et al., 1999; Jenne et al., 2000; Deng et al.,2006; Li et al., 2005; ref from Deng et al., J VI 2006 paper) and thusadds to the immunosuppressive and immunoevasive effects of tumorsthemselves.

Poxviruses however are extraordinarily adept at evading and antagonizingmultiple innate immune signaling pathways by encoding proteins thatinterdict the extracellular and intracellular components of thosepathways (Seet et al. Annu. Rev. Immunol. 21377-423 (2003)). Chief amongthe poxvirus antagonists of intracellular innate immune signaling is thevaccinia virus duel Z-DNA and dsRNA-binding protein E3, which caninhibit the PKR and NF-κB pathways (Cheng et al. Proc. Natl. Acad. Sci.USA 894825-4829 (1992); Deng et al. J. Virol. 809977-9987 (2006)) thatwould otherwise be activated by vaccinia virus infection. A mutantvaccinia virus lacking the E3L gene (ΔE3L) has a restricted host range,is highly sensitive to IFN, and has greatly reduced virulence in animalmodels of lethal poxvirus infection (Beattie et al. Virus Genes. 1289-94(1996); Brandt et al. Virology 333263-270 (2004)). Recent studies haveshown that infection of cultured cell lines with ΔE3L virus elicitsproinflammatory responses that are masked during infection withwild-type vaccinia virus (Deng et al. J. Virol. 809977-9987 (2006);Langland et al. J. Virol. 8010083-10095). The inventors have reportedthat infection of a mouse epidermal dendritic cell line with wild-typevaccinia virus attenuated proinflammatory responses to the TLR agonistslipopolysaccharide (LPS) and poly(I:C), an effect that was diminished bydeletion of E3L. Moreover, infection of the dendritic cells with ΔE3Lvirus triggered NF-κB activation in the absence of exogenous agonists(Deng et al. J. Virol. 809977-9987 (2006)). The inventors of the presentdisclosure have also showed that whereas wild-type vaccinia virusinfection of murine keratinocytes does not induce the production ofproinflammatory cytokines and chemokines, infection with ΔE3L virus doesinduce the production of IFN-β, IL-6, CCL4 and CCL5 from murinekeratinocytes, which is dependent on the cytosolic dsRNA-sensing pathwaymediated by the mitochondrial antiviral signaling protein (MAVS; anadaptor for the cytosolic RNA sensors RIG-I and MDA5) and thetranscription factor IRF3 (Deng et al., J Virol. 2008 November; 82(21):10735-10746.). See also international Application PCT/US2016/019663filed by the inventor and co-workers on Feb. 25, 2016; and provisionalapplication No. 62/149,484 filed on Apr. 17, 2015 and its correspondinginternational application, PCT/US2016/028184. These applications areherein incorporated by reference in their entirety.

E3LΔ83N virus with deletion of the Z-DNA-binding domain is 1,000-foldmore attenuated than wild-type vaccinia virus in an intranasal infectionmodel (Brandt et al., 2001). E3LΔ83N also has reduced neurovirulencecompared with wild-type vaccinia in an intra-cranial inoculation model(Brandt et al., 2005). A mutation within the Z-DNA binding domain of E3(Y48A) resulting in decreased Z-DNA-binding leads to decreasedneurovirulence (Kim et al., 2003). Although the N-terminal Z-DNA bindingdomain of E3 is important in viral pathogenesis, how it affects hostinnate immune sensing of vaccinia virus is not well understood. Theinventors have previously shown that myxoma virus but not wild-typevaccinia infection of murine plasmacytoid dendritic cells induces type IIFN production via the TLR9/MyD88/IRF5/IRF7-dependent pathway (Dai etal., 2011). Myxoma virus E3 ortholog M029 retains the dsRNA-bindingdomain of E3 but lacks the Z-DNA binding domain of E3. It was found thatthe Z-DNA-binding domain of E3 (but probably not Z-DNA-binding activityper se) plays an important role in inhibiting poxviral sensing in murineand human pDCs (Dai et al., 2011; Cao et al., 2012).

Deletion of E3L sensitizes vaccinia virus replication to IFN inhibitionin permissive RK13 cells and results in a host range phenotype, wherebyΔE3L cannot replicate in HeLa or BSC40 cells (Chang et al., 1995). TheC-terminal dsRNA-binding domain of E3 is responsible for the host rangeeffects, whereas E3LΔ83N virus with deletion of the N-terminalZ-DNA-binding domain is replication competent in HeLa and BSC40 cells(Brandt et al., 2001). Because E3LΔ83N is 1000-fold more attenuated thanwild-type vaccinia, in this application, the inventors explored its useas an attenuated replication competent vaccinia viral vector for furtherconstruction of immune-stimulating immunotherapeutic agent againstvarious cancers.

Vaccinia virus (Western Reserve strain; WR) with deletion of thymidinekinase is highly attenuated in non-dividing cells but is replicative intransformed cells (Buller et al., 1988). TK-deleted vaccinia virusselectively replicates in tumor cells in vivo (Puhlmann et al., 2000).Thorne et al. showed that compared with other vaccinia strains, WRstrain has the highest burst ratio in tumor cell lines relative tonormal cells (Thorne et al., 2007). The inventors selected a derivativeof this strain, vaccinia E3LΔ83N WR strain as their vector for furthermodification.

Human Flt3L (Fms-like tyrosine kinase 3 ligand), a type I transmembraneprotein that stimulates the proliferation of bone marrow cells, wascloned for in 1994 (Lyman et al., 1994). The use of hFlt3L has beenexplored in various preclinical and clinical settings including stemcell mobilization in preparation for bone marrow transplantation, cancerimmunotherapy such as expansion of dendritic cells, as well as anvaccine adjuvant. Recombinant human Flt3L (rhuFlt3L) has been tested inmore than 500 human subjects and is bioactive, safe, and well tolerated(Fong et al., 1998; Maraskovsky et al., 2000; Shackleton et al., 2004;He et al., 2014; Anandasabapathy et al., 2015). Much progress has beenrecently made in the understanding of the critical role of the growthfactor Flt3L in the development of DC subsets, including CD8α⁺/CD103⁺DCs and pDCs (McKenna et al., 2000; Waskow et al., 2008; Liu et al.,2007; 2009; Naik et al., 2006; Ginhoux et al., 2009).

SUMMARY OF THE DISCLOSURE

In the present disclosure, the inventors generated recombinantE3LΔ83N-TK⁻ virus and also built a construct of the same virusexpressing human Flt3L, with the goal of delivering this growth factorto the tumor microenvironment to facilitate recruitment, differentiationand function of immune cells, including CD103⁺/CD8α dendritic cells(DCs). A similar goal was pursued with E3LΔ83N-TK⁻ expressing GM-CSF.However, experiments were also conducted with “naked” E3LΔ83N-TK⁻ andwith inactivated (specifically heat-inactivated) virus and viralconstructs with favorable results, especially when administered inconjunction with checkpoint blockade inhibition therapy.

The present disclosure concerns methods and compositions for thetreatment of solid tumors using a replication competent attenuatedvaccinia virus either alone or in combination with immune checkpointblocking agents. In some embodiments, methods and compositions involvedeletion of Z-DNA-binding domain of E3Land thymidine kinase (TK) genefrom wild-type vaccinia (Western Reserve strain, WR) and the expressionof GM-CSF or Flt3L under vaccinia promoter.

This invention relates to the discovery that E3LΔ83N-TK⁻ is attenuatedin vitro and in vivo, and therefore it is a safer oncolytic viruscompared with vaccinia comprising only TK deletion. RecombinantE3LΔ83N-TK− viruses expressing either GM-CSF or Flt3L may have an addedbenefit of immune stimulation. Infection by these viruses induces cancercell death, which leads to tumor antigen release. Intratumoral injectionof E3LΔ83N-TK⁻ (VC-TK⁻), E3LΔ83N-TK⁻-mGM-CSF (VC-TK⁻-mGM-CSF),E3LΔ83N-TK⁻-hFlt3L(VC-TK⁻-hFlt3L) leads to tumor regression anderadication of the injected tumor, and to the generation of antitumoralimmunity. In addition, the combination of intratumoral delivery ofeither E3LΔ83N-TK⁻-mGM-CSF or E3LΔ83N-TK⁻-hFlt3L with immune checkpointblocking agent dramatically improved survival (both number survived andduration) compared with virotherapy alone. Finally, intratumoraldelivery of inactivated E3LΔ83N-TK⁻-mGM-CSF after heating the virus at55° C. (Heat-VC-TK⁻-mGM-CSF) leads to more efficient tumor eradicationat the contralateral non-injected site than the live virus. It ispossible that alternating intratumoral delivery of the live virus withthe inactivated virus might achieve better efficacies than either agentalone.

Therefore, E3LΔ83N-TK⁻, E3LΔ83N-TK⁻-GM-CSF, E3LΔ83N-TK⁻-hFlt3L viruses,replicative or inactivated, can be used as oncolytic therapy andimmunotherapy for the treatment of solid tumors. (It is understood thathuman GM-CSF would be used in viral constructs for human use. Results inmice with mouse GM-CSF are relevant to the human application of thepresent substances and compositions as the animal models used herein arewell-accepted.) Additionally, the inventors of the present disclosurehave shown that the combination of intratumoral delivery of oncolyticvirus with immune checkpoint blocking agent leads to more efficienttumor eradication and better survival than either agent alone.

The recombinant vaccinia viruses can be administered intratumorally,intravenously, intraperitoneally, or intracranially or via a combinationof localized (e.g., intratumoral) injection and a systemic or in anyevent more diffuse injection. The localized (e.g., intratumoral)injection of viruses can be used for various stages of tumors. For earlystage cancer, virotherapy can be used 2-3 weeks prior to surgicalremoval of the tumor. During that time frame, the host would havedeveloped systemic anti-tumor adaptive immunity. For advanced cancer,virotherapy can be used in combination with other treatment modalities,including surgery, chemotherapy, targeted therapy, radiation, and immunecheckpoint therapy, which will be detailed below.

The present inventors hypothesized that intratumoral injection of one ormore of E3LΔ83N-TK⁻, E3LΔ83N-TK⁻-GM-CSF, and E3LΔ83N-TK⁻-hFlt3L viruseswould provide additional beneficial effects to a PD-1 or CTLA-4targeting approach, through altering tumor immune suppressiveenvironment via the activation of immune cells including dendritic cellsand macrophages, as well as facilitating tumor antigen presentation.Indeed, it was observed that treatment with a combination ofVC-TK⁻-GM-CSF and a checkpoint blockade inhibitor leads to developmentof immunity against a subsequent challenge with heterologous tumor.Similar results were observed with heat-inactivated viral constructcombined or, surprisingly, even when not combined with immune checkpointblockade therapy.

In further studies, the antitumor immunity of the TK⁻ virus and to agreater extent the viral-hFlt3L construct was found to includeactivation of effector CD8⁺ and CD4⁺ T cells (with the construct beingmore effective), leading to the expectation that similar resultsqualitatively would be observed after injection of viral GM-CSFconstruct. Further, the antitumor immunity of the viral-hFlt3L constructalso led to an increase of CD103⁺ dendritic cells.

The foregoing antitumor results are not limited to melanoma but extendto other solid tumors such as breast cancer and colon carcinoma.Interestingly, certain viruses are more effective in one type of cancerand other viruses are more effective in another. Thus the use of thepresent therapies is subject to optimization. However, this does notdetract from the utility of the present therapies, all the more becausein cancer response to most therapeutic modalities is subject tocase-by-case variability depending on differences in disease, thepresence or absence of tumor infiltrating immune cells, in genetic andepigenetic factors, and in the use or nonuse of prior therapies.Furthermore, GM-CSF and FLt3L viral constructs have shown, or in lightof the aforedescribed studies, are expected to show efficacy againstestablished tumor models, which model advanced stage tumors.

In one aspect, the disclosure is directed to methods for treating asolid malignant tumor in a subject comprising delivering to tumor cellsof the subject an amount of one or more of E3LΔ83N-TK⁻,E3LΔ83N-TK⁻-GM-CSF, and E3LΔ83N-TK⁻-hFlt3L viruses and viral constructs,including replicative (live) and inactivated versions thereof, effectiveto induce the immune system of the subject to mount an immune responseagainst the tumor, for example as set forth above in this Summary so asto accomplish one or more of the following (regardless of order): reducethe size of the tumor, eradicate the tumor, inhibit growth of the tumor,or inhibit metastasis or metastatic growth of the tumor and therebytreat the tumor.

In another aspect, the disclosure is directed to an active substanceselected from the group consisting of E3LΔ83N-TK⁻, E3LΔ83N-TK⁻-GM-CSF,and E3LΔ83N-TK⁻-hFlt3L, in replicative or inactivated form. Thesesubstances are each useful as sole active ingredients or in combinationwith two or more of them and optionally in combination with othertherapeutic modalities, in an amount or in amounts effective to treat asolid malignant tumor or to elicit in the treated subject an immuneresponse against the tumor, upon local administration to the tumor. Theimmune response may include one or more of the following immunologicaleffects

-   -   oncolysis of tumor cells and release of tumor antigen;    -   an increase in cytotoxic CD8⁺ T cells within the tumor and/or in        tumor-draining lymph nodes;    -   induction of maturation of dendritic cells infiltrating said        tumor or circulating in remote locations within the patient's        body through induction of type I IFN;    -   induction of effector CD4⁺ T cells in the subject recognizing        tumor cells within the tumor and/or in tumor draining lymph        nodes;

In a related aspect, the disclosure is directed to compositionscomprising an effective amount for treating a solid malignant tumor orfor eliciting in a patient an immune response against the tumor anactive ingredient comprising one or more of E3LΔ83N-TK⁻,E3LΔ83N-TK⁻-GM-CSF, and E3LΔ83N-TK⁻-hFlt3L viruses and viral constructs,including replicative and inactivated versions thereof and apharmaceutically acceptable excipient.

In another aspect, the disclosure is directed to a method for treating amalignant tumor comprising:

-   -   delivering to tumor cells of the subject an amount of live or        inactivated one or more of E3LΔ83N-TK⁻, E3LΔ83N-TK⁻-GM-CSF, and        E3LΔ83N-TK⁻-hFlt3L viruses effective to induce the immune system        of the subject to mount an immune response against the tumor.

In some embodiments one or more of the following specific features arealso present:

-   -   the recruitment and activation of effector CD4⁺ and CD8⁺ T cells        is accompanied;    -   the tumor is melanoma or colon carcinoma or breast carcinoma;    -   a regimen of periodic delivery of one or more of E3LΔ83N-TK⁻,        E3LΔ83N-TK⁻-GM-CSF, and E3LΔ83N-TK⁻-hFlt3L viruses is continued        until it induces tumor regression or eradication;    -   a regimen of periodic delivery of one or more of E3LΔ83N-TK⁻,        E3LΔ83N-TK⁻-GM-CSF, and E3LΔ83N-TK⁻-hFlt3L viruses is continued        for several weeks, months or years or indefinitely as long as        benefits persist;    -   a regimen of periodic delivery of one or more of E3LΔ83N-TK⁻,        E3LΔ83N-TK⁻-GM-CSF, and E3LΔ83N-TK⁻-hFlt3L viruses is continued        indefinitely until the maximum tolerated dose is reached;    -   delivery of one or more of E3LΔ83N-TK⁻, E3LΔ83N-TK⁻-GM-CSF, and        E3LΔ83N-TK⁻-hFlt3L viruses is by parenteral injection;    -   delivery of one or more of E3LΔ83N-TK⁻, E3LΔ83N-TK⁻-GM-CSF, and        E3LΔ83N-TK⁻-hFlt3L viruses is by intratumoral injection;    -   delivery of the one or more of E3LΔ83N-TK⁻, E3LΔ83N-TK⁻-GM-CSF,        and E3LΔ83N-TK⁻-hFlt3L viruses is by intravenous injection;    -   the subject is a human;    -   E3L≢83N-TK⁻, E3LΔ83N-TK⁻-GM-CSF, and/or E3LΔ83N-TK⁻-hFlt3L        viruses is delivered at a dosage per administration within the        range of about 10⁵-10¹⁰ plaque-forming units (pfu);    -   E3LΔ83N-TK⁻, E3LΔ83N-TK⁻-GM-CSF, and/or E3LΔ83N-TK⁻-hFlt3L        viruses is delivered at a dosage per administration within the        range of about 10⁶ to about 10⁹ plaque-forming units (pfu);    -   the amount delivered is sufficient to infect all tumor cells;    -   the delivery is repeated with a frequency within the range from        once per month to two times per week;    -   the treatment continues for a period of weeks, months or years;    -   the delivery is repeated with a frequency within the range from        once per month to two times per week;    -   the melanoma is metastatic melanoma.

Delivery of E3LΔ83N-TK⁻, E3LΔ83N-TK⁻-GM-CSF, E3LΔ83N-TK⁻-hFlt3L virusesin the locale of the tumor induces the immune system of a subjectafflicted with a malignant solid tumor to mount an immune responseagainst the tumor. Stimulation of the subject's immune system againstthe tumor can be manifest (and may indeed be tested) by one or more ofthe following immunological effects

-   -   oncolysis of tumor cells and release of tumor antigen;    -   an increase in cytotoxic CD8⁺ T cells within the tumor and/or in        tumor-draining lymph nodes;    -   oncolysis and release of tumor antigens;    -   induction of effector T cells in the subject recognizing tumor        cells within the tumor and/or in tumor draining lymph nodes.

In certain embodiments, present invention relates to an isolated andpurified active substance comprising E3LΔ83N-TK⁻-hFlt3L in replicativeor inactivated form suitable for use as an immunotherapeutic agentagainst a malignant solid tumor.

In yet further aspects, the invention relates to a method for treating asubject afflicted with one or more solid malignant tumors, the methodcomprising delivering to cells of the tumor replication competent orinactivated E3LΔ83N-TK⁻-hFlt3L virus and thereby treating the tumor.

In certain embodiments, the amount is effective to accomplish one ormore of the following:

-   -   a. induce the immune system of the subject to mount an immune        response against the tumor;    -   b. reduce the size of the tumor;    -   c. eradicate the tumor;    -   d. inhibit growth of the tumor;    -   e. inhibit metastasis of the tumor; and    -   f. reduce or eradicate metastatic tumor.

In other embodiments, the tumor includes tumor located at the site ofdelivery, or tumor located both at said site and elsewhere in the bodyof the subject.

In yet further embodiments, the immune response comprises one or more ofthe following:

-   -   a. oncolysis of tumor cells and release of tumor antigen;    -   b. increase in cytotoxic CD8⁺ T cells within the tumor and/or in        tumor-draining lymph nodes;    -   c. induction of maturation of dendritic cells infiltrating said        tumor through induction of type I IFN;    -   d. induction of activated CD4⁺ effector T cells in the subject        recognizing tumor cells within the tumor or systemically.

In additional embodiments, the tumor is primary or metastatic melanomaor breast carcinoma or colon carcinoma.

In yet additional aspects the invention relates to a method for treatinga solid malignant tumor in a subject comprising delivering to tumorcells of the subject an amount of replication competent or inactivatedE3LΔ83N-TK⁻-hFlt3L virus effective to induce the immune system of thesubject to mount an immune response against the tumor.

In certain embodiments, the immune response is systemic.

In additional embodiments, the immune response effects or contributes toone or more of the following: reduction of the size of the tumor,eradication of the tumor, inhibition of tumor or metastatic growth.

In further embodiments, the virus is effective to accomplish one or moreof the following:

a. induce the immune system of the subject to mount an immune responseagainst the tumor;

-   -   g. b. reduce the size of the tumor;    -   h. c eradicate the tumor;    -   i. d. inhibit growth of the tumor;    -   j. e. inhibit metastasis of the tumor; and        reduce or eradicate metastatic tumor.

In yet additional aspects the invention relates to method for treating amalignant tumor in a subject, the method comprising delivering to tumorcells of the subject replication competent or inactivatedE3LΔ83N-TK−-hFlt3L virus in an amount effective to induce the immunesystem of the subject to mount an immune response against the tumor andconjointly administering or having administered to the subject a secondamount of an immune checkpoint blocking agent effective to block immunesuppressive mechanisms within the tumor elicited by tumor cells, stromalcells, or tumor infiltrating immune cells.

In yet additional aspects the invention relates to a method for treatinga malignant tumor in a subject, the method comprising delivering orhaving delivered to tumor cells of the subject replication competent orinactivated E3LΔ83N-TK−-hFlt3L virus in an amount effective to inducethe immune system of the subject to mount an immune response against thetumor and conjointly administering or having administered to the subjecta second amount of an immune checkpoint blocking agent effective toblock immune suppressive mechanisms within the tumor elicited by tumorcells, stromal cells, or tumor infiltrating immune cells.

In certain embodiments, the conjoint administration is effective toaccomplish one or more of the following:

-   -   a. induce the immune system of the subject to mount an immune        response against the tumor;    -   b. reduce the size of the tumor;    -   c. eradicate the tumor;    -   d. inhibit growth of the tumor;    -   e. inhibit metastasis of the tumor; and    -   f. reduce or eradicate metastatic tumor.

In yet additional aspects the tumor is primary or metastatic malignantmelanoma or breast carcinoma or colon carcinoma. In yet additionalaspects the virus is heat-inactivated.

In yet additional aspects the invention relates to a compositioncomprising an effective amount for treating a patient afflicted with asolid malignant tumor an active ingredient comprisingE3LΔ83N-TK⁻-hFlt3L, in replicative or inactivated form, or both, and apharmaceutically acceptable excipient.

In yet additional aspects the amount is effective to accomplish one ormore of the following: reduce the size of the tumor, eradicate thetumor, inhibit growth of the tumor, or inhibit metastasis or metastaticgrowth of the tumor and thereby treat the tumor.

In yet additional aspects the amount is effective to elicit in thetreated subject an immune response against the tumor and any metastasesthereof, upon local delivery to tumor cells of the subject.

In yet additional aspects the immune response includes one or more ofthe following:

-   -   an increase in cytotoxic CD8⁺ T cells within the tumor and/or in        tumor-draining lymph nodes;    -   induction of maturation of dendritic cells infiltrating said        tumor or circulating in remote locations within the patient's        body through induction of type I IFN;    -   induction of effector CD4⁺ T cells in the subject recognizing        tumor cells within the tumor and/or in tumor draining lymph        nodes.

In yet additional aspects the invention relates to an isolated purifiedactive substance selected from the group consisting of E3LΔ83N-TK⁻,E3LΔ83N-TK⁻-GM-CSF, and E3LΔ83N-TK⁻-Flt3L, in replicative or inactivatedform, suitable for use as a immunotherapeutic agent against a malignantsolid tumor.

In yet additional aspects the invention relates to a compositioncomprising an effective amount for treating a patient afflicted with asolid malignant tumor of an active ingredient comprising one or more ofE3LΔ83N-TK⁻, E3LΔ83N-TK⁻-GM-CSF, and E3LΔ83N-TK⁻-hFlt3L viruses andviral constructs, each optionally in replicative or inactivated form,and a pharmaceutically acceptable excipient.

In yet additional aspects the composition contains two or more of saidviruses and viral constructs.

In yet additional aspects the amount is effective to accomplish one ormore of the following: reduce the size of the tumor, eradicate thetumor, inhibit growth of the tumor, or inhibit metastasis or metastaticgrowth of the tumor and thereby treat the tumor.

In yet additional aspects the amount is effective to elicit in thetreated subject an immune response against the tumor and other tumors inthe treated subject's body, upon local delivery to tumor cells of thesubject.

In yet additional aspects the immune response may include one or more ofthe following:

-   -   an increase in cytotoxic CD8⁺ T cells within the tumor and/or in        tumor-draining lymph nodes;    -   induction of maturation of dendritic cells infiltrating said        tumor or circulating in remote locations within the patient's        body through induction of type I IFN;    -   induction of effector CD4⁺ T cells in the subject recognizing        tumor cells within the tumor and/or in tumor draining lymph        nodes.

In yet additional aspects the invention relates to a method for treatinga solid malignant tumor in a subject comprising delivering to tumorcells of the subject an amount of one or more of E3LΔ83N-TK⁻,E3LΔ83N-TK⁻-GM-CSF, and E3LΔ83N-TK⁻-hFlt3L viruses and viral constructs,each optionally in replicative or inactivated form, effective to inducethe immune system of the subject to mount an immune response against thetumor.

In yet additional aspects the invention relates to a method for treatinga solid malignant tumor in a subject comprising delivering to tumorcells of the subject an amount of one or more of E3LΔ83N-TK⁻,E3LΔ83N-TK⁻-GM-CSF, and E3LΔ83N-TK⁻-hFlt3L viruses and viral constructs,including replicative and inactivated versions of each of the foregoing,effective to accomplish one or more of the following (regardless oforder):

-   -   a. induce the immune system of the subject to mount an immune        response against the tumor;    -   b. reduce the size of the tumor;    -   c. eradicate the tumor;    -   d. inhibit growth of the tumor;    -   e. inhibit metastasis of the tumor; and    -   f. reduce or eradicate metastatic tumor.

In yet additional aspects the immune response may include one or more ofthe following immunological effects

-   -   an increase in cytotoxic CD8⁺ T cells within the tumor and/or in        tumor-draining lymph nodes;    -   induction of maturation of dendritic cells infiltrating said        tumor or circulating in remote locations within the patient's        body through induction of type I IFN;    -   induction of effector T cells in the subject recognizing tumor        cells within the tumor and/or in tumor draining lymph nodes.

In yet additional aspects the invention relates to a method for treatinga malignant tumor in a subject, the method comprising delivering orhaving delivered to the subject tumor cells of the subject replicationcompetent or inactivated E3LΔ83N-TK⁻, E3LΔ83N-TK⁻-GM-CSF, andE3LΔ83N-TK⁻-hFlt3L viruses or viral constructs, each optionally inreplicative or inactivated form, in an amount effective to induce theimmune system of the subject to mount an immune response against thetumor and conjointly administering or having administered to the subjecta second amount of an immune checkpoint blocking agent effective toblock immune suppressive mechanisms within the tumor elicited by tumorcells, stromal cells, or tumor infiltrating immune cells.

In yet additional aspects the conjoint administration is effective toaccomplish one or more of the following:

-   -   a) induce the immune system of the subject to mount an immune        response against the tumor;    -   b) reduce the size of the tumor;    -   c) eradicate the tumor;    -   d) inhibit growth of the tumor;    -   e) inhibit metastasis of the tumor; and reduce or eradicate        metastatic tumor.

In yet additional aspects the invention relates to a method for treatinga malignant tumor in a subject, wherein the subject has been previouslytreated or dosed with replication competent or inactivated E3LΔ83N-TK⁻,E3LΔ83N-TK⁻-GM-CSF, and E3LΔ83N-TK⁻-hFlt3L viruses or viral constructs,each optionally in replicative or inactivated form, in an amounteffective to induce the immune system of the subject to mount an immuneresponse against the tumor

In yet additional embodiments, the method comprises delivering to thesubject tumor cells of the subject an amount of an immune checkpointblocking agent effective to block immune suppressive mechanisms withinthe tumor elicited by tumor cells, stromal cells, or tumor infiltratingimmune cells.

In yet additional aspects the invention relates to a method for treatinga malignant tumor in a subject, wherein the subject has been previouslytreated or dosed with an amount of an immune checkpoint blocking agenteffective to block immune suppressive mechanisms within the tumorelicited by tumor cells, stromal cells, or tumor infiltrating immunecells

In yet additional embodiments, the method comprises delivering or havingdelivered to the subject tumor cells of the subject replicationcompetent or inactivated E3LΔ83N-TK⁻, E3LΔ83N-TK⁻-GM-CSF, andE3LΔ83N-TK⁻-hFlt3L viruses or viral constructs, each optionally inreplicative or inactivated form, in an amount effective to induce theimmune system of the subject to mount an immune response against thetumor.

In yet additional aspects the immune checkpoint blocking agent comprisesCTLA-4, CD80, CD86, PD-1, PDL1, PDL2, LAG3, B7-H3, B7-H4, TIM3, ICOS, IIDLBCL inhibitors, BTLA, or any combination thereof.

In yet additional aspects the immune checkpoint blocking agent comprisesipilimumab, nivolumab, pembrolizumab, pidilizumab, AMP-224, MPDL3280A,BMS-936559, MEDI4736, MSB 00107180, or any combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-D is a series of graphs showing a one-step growth of E3LΔ83N(VC) and ΔE3L (VI) vaccinia viruses in murine and human melanoma celllines. Murine B16-F10 melanoma cells and human melanoma cells SK-MEL39,SK-MEL188, and SK-MEL90 were infected with either E3LΔ83N (VC) or ΔF3L(VI) at a MOI of 5. Cells were collected at various times post infectionand viral yields (log pfu) were determined.

FIG. 2 is a schematic diagram of homologous recombination betweenplasmid DNA and viral genomic DNA at the thymidine kinase (TK) locus.pCB plasmid was used to insert specific gene of interest (SG), in thiscase, murine GM-CSF (mGM-CSF), and human Flt3L (hFlt3L) under thecontrol of the vaccinia synthetic early and late promoter (Pse/l). TheE. coli xanthine-guanine phosphoribosyl transferase gene (gpt) under thecontrol of vaccinia P7.5 promoter was used as a drug selection marker.These two expression cassettes were flanked by partial sequence of TKgene (TK-L and TK-R) on each side. The plasmid DNA lacking SG was usedas a vector control. Homologous recombination that occurred at the TKlocus of the plasmid DNA and VC genomic DNA results in the insertion ofSG and gpt expression cassettes or gpt alone into the VC genomic DNA togenerate VC-TK⁻-mGM-CSF, VC-TK⁻-hFlt3L, or VC-TK⁻. The recombinantviruses were enriched in the presence of gpt selection medium includingMPA, xanthine and hypoxanthine, and plaque purified in the presence ofthe drug selection medium for 4-5 rounds until the appropriaterecombinant viruses without contaminating VC were obtained.

FIG. 3 is an image of PCR analysis of recombinant viruses, showingsuccessful generation of VC-TK⁻, VC-TK⁻-mGM-CSF, and VC-TK⁻-hFlt3L. VCrecombinant viruses genomic DNAs were analyzed by PCR to verify theinsertions and to make sure there were no contaminating patent virusparticles (VC).

FIG. 4A-B show Western blot results. FIG. 4A shows a Western blotanalysis of mGM-CSF expression in VC-TK⁻-mGM-CSF-infected murine B16melanoma cells and human SK-MEL-28 melanoma cells. FIG. 4A shows datafrom B16-F10 and SK-MEL-28 cells that were infected or mock infectedwith VC-TK⁻-mGM-CSF. Cell lysates and supernatants were collected atvarious times post infection. Western blot analyses were performed usinganti-mGM-CSF antibody and anti-GAPDH as a loading control. FIG. 4B showsa Western blot analysis of hFlt3L expression in VC-TK⁻-hFlt3L-infectedmurine and human melanoma cells. B16-F10, SK-MEL-146, and SK-MEL-28cells were infected or mock infected with VC-TK⁻-hFlt3L. Cell lysateswere collected at various times post infection. Western blot analysis ofcell lysates was performed using anti-hFlt3L antibody and anti-GAPDH asa control.

FIG. 5A-B shows a one-step growth of VC, VC-TK⁻, VC-TK⁻-mGM-CSF, andVC-TK⁻-hFlt3L in B16-F10 melanoma cells. B16-F10 melanoma cells wereinfected with VC, VC-TK⁻, VC-TK⁻-mGM-CSF, or VC-TK⁻-hFlt3L at a MOI of0.1. Cells were collected at various times post infection and viralyields were determined by titrating on BSC40 cells. Viral yields (logpfu) were plotted against hours post infection in (FIG. 5A). The foldchanges of viral yields at 72 h over those at 1 h post infection wereplotted in (FIG. 5B).

FIG. 6 is a scheme of treatment plan in which B16-F10 melanomas weretreated with intratumoral injection of viruses in the presence orabsence of immune checkpoint blockade. Briefly, B16-F10 melanoma cellswere implanted intradermally to left and right flanks of C57B/6 mice(5×10⁵ cells to the right flank and 1×10⁵ cells to the left flank). 7days post tumor implantation, the mice were treated with intratumoralinjections of viruses twice a week with or without intraperitonealdelivery of immune checkpoint blockade antibodies. We measured tumorsizes and monitored survival in the next 8 weeks.

FIG. 7A-L shows a series of graphical representations of intratumoralinjection of VC-TK⁻, VC-TK⁻-mGM-CSF, or VC-TK⁻-hFlt3L alone or incombination with intraperitoneal delivery of anti-CTLA-4 antibody in aB16-F10 melanoma bilateral implantation model. B16-F10 melanoma cells(5×10⁵) were implanted intradermally into the shaved skin on the rightflank, and (1×10⁵) cells were implanted to the left flank. At 7 dayspost implantation, the right side tumors (about 3 mm in diameter) wereinjected twice weekly with either PBS, VC-TK⁻, VC-TK⁻-mGM-CSF, orVC-TK⁻-hFlt3L (2×10⁷ pfu). Some groups of mice were treated with acombination of intratumoral delivery of either VC-TK⁻-mGM-CSF, orVC-TK⁻-hFlt3L (2×10⁷ pfu) and intraperitoneal delivery of anti-CTLA-4antibody (100 μg/mouse) twice weekly. Tumor sizes were measured andmouse survival was monitored over time. (FIG. 7A, B) graphs of volume ofinjected tumors (FIG. 7A) and non-injected tumors (FIG. 7B) at variousdays post injection with PBS (n=10). (FIG. 7C, D) graphs of volume ofinjected tumors (FIG. 7C) and non-injected tumors (FIG. 7D) at variousdays post injection with VC-TK⁻ (n=10). (FIG. 7E, F) graphs of volume ofinjected tumors (FIG. 7E) and non-injected tumors (FIG. 7F) at variousdays post injection with VC-TK⁻-mGM-CSF (n=10). (FIG. 7G, H) graphs ofvolume of injected tumors (FIG. 7G) and non-injected tumors (FIG. 7H) atvarious days post injection with VC-TK⁻-hFlt3L. (FIG. 7I, J) graphs ofvolume of injected tumors (FIG. 7I) and non-injected tumors (FIG. 7J) atvarious days post injection with VC-TK⁻-mGM-CSF with intraperitonealdelivery of anti-CTLA-4 antibody (n=10). (FIG. 7K, L) graphs of volumeof injected tumors (FIG. 7K) and non-injected tumors (FIG. 7L) atvarious days post injection with VC-TK⁻-hFlt3L with intraperitonealdelivery of anti-CTLA-4 antibody. (FIG. 7M) Kaplan-Meier survival curveof mice treated with PBS, VC-TK⁻, VC-TK⁻-mGM-CSF, VC-TK⁻-hFlt3L,VC-TK⁻-mGM-CSF+anti-CTLA-4, or VC-TK⁻-hFlt3L+anti-CTLA-4. Survival datawere analyzed by log-rank (Mantel-Cox) test. *, P<0.05; ****, P<0.0001.

FIG. 8A-O shows a series of graphical representations of intratumoraldelivery of live VC-TK⁻-mGM-CSF, Heat-inactivated VC-TK⁻-mGM-CSF, liveplus Heat-inactivated E3LΔ83N-TK⁻-mGM-CSF with or withoutintraperitoneal delivery anti-PD-L1 antibody in a B16-F10 melanomabilateral implantation model. B16-F10 melanoma cells were implantedbilaterally as described above in FIG. 6. At 7 days post implantation,the right side tumors (about 3 mm in diameter) were injected with eitherPBS, live VC-TK⁻-mGM-CSF (2×10⁷ pfu), Heat-inactivated VC-TK⁻-mGM-CSF(an equivalent of 2×10⁷ pfu), live (1×10⁷ pfu) plus Heat-inactivatedVC-TK⁻-mGM-CSF (an equivalent of 1×10⁷ pfu), with or withoutintraperitoneal delivery of anti-PD-L1 antibody (200 μg/mouse) twiceweekly. Tumor size was measured and mouse survival was monitored overtime. FIG. 8A-B are graphs showing the volume of injected tumors (FIG.8A) and non-injected tumors (FIG. 8B) at various days post injectionwith PBS (n=5). FIGS. 8 C, D are graphs showing the volume of injectedtumors (FIG. 8C) and non-injected tumors (FIG. 8D) at various days postinjection with VC-TK⁻-mGM-CSF (n=9). (FIG. 8E, F) are graphs showing thevolume of injected tumors (FIG. 8E) and non-injected tumors (FIG. 8F) atvarious days post injection with Heat-inactivated VC-TK⁻-mGM-CSF (n=9).FIG. 8G, H are graphs showing the volume of injected tumors (FIG. 8G)and non-injected tumors (FIG. 8H) at various days post injection withlive+Heat-inactivated VC-TK⁻-mGM-CSF (n=9). FIGS. 8 I, J are graphsshowing the volume of injected tumors (FIG. 8I) and non-injected tumors(FIG. 8J) at various days post injection with VC-TK⁻-mGM-CSF in thepresence of systemic delivery of anti-PD-L1 antibody (n=9). FIG. 8K, Lare graphs showing the volume of injected tumors (FIG. 8K) andnon-injected tumors (FIG. 8L) at various days post injection withHeat-inactivated VC-TK⁻-mGM-CSF in the presence of systemic delivery ofanti-PD-L1 antibody (n=9). FIGS. 8 M, N are graphs showing the volume ofinjected tumors (FIG. 8M) and non-injected tumors (FIG. 8N) at variousdays post injection with live+inactivated VC-TK⁻-mGM-CSF in the presenceof systemic delivery of anti-PD-L1 antibody (n=9). FIG. 8O is aKaplan-Meier survival curve of mice treated with PBS, liveVC-TK⁻-mGM-CSF, Heat-inactivated VC-TK⁻-mGM-CSF, live+Heat-inactivatedVC-TK⁻-mGM-CSF, VC-TK⁻-mGM-CSF+anti-PD-L1, Heat-inactivatedVC-TK⁻-mGM-CSF+anti-PD-L1, or live+Heat-inactivatedVC-TK⁻-mGM-CSF+anti-PD-L1 antibody. Survival data were analyzed bylog-rank (Mantel-Cox) test. *, P<0.05; ****, P<0.0001.

FIG. 9 shows a Kaplan-Meier survival curve of mice after tumorrechallenge with heterologous tumor MC38. These mice had initiallytreated with the following regimen for B16-F10 melanoma and surved.These agents include live VC-TK⁻-mGM-CSF, Heat-inactivatedVC-TK⁻-mGM-CSF, live+Heat-inactivated VC-TK⁻-mGM-CSF,VC-TK⁻-mGM-CSF+anti-PD-L1, Heat-inactivated VC-TK⁻-mGM-CSF+anti-PD-L1,or live+Heat-inactivated VC-TK⁻-mGM-CSF+anti-PD-L1 antibody. A group ofnaïve mice that have never been exposed either to viruses or tumors wereused as controls. MC38 (1×10⁵ cells) were implanted intradermally. Tumorgrowth and mice survival were monitored closely.

FIG. 10A-D is a series of graphical representations of data collectedafter intratumoral injection of VC-TK− or VC-TK⁻-hFlt3L which shows thatVC-TK⁻-hFlt3L is more effective than VC-TK⁻ virus in activating bothCD8⁺ and CD4⁺ T cells in both injected and non-injected tumors in abilateral melanoma model. FIG. 10A consists of representative flowcytometry dot plots of CD8⁺Granzyme B⁺ cells in injected (right plot)and non-injected (left plot) tumors of mice treated variously with PBS,VC-TK⁻, or VC-TK⁻-hFlt3L. FIG. 10B consists of dot plots of CD4⁺GranzymeB⁺ cells in injected (right plot) and non-injected (left plot) tumors ofmice treated variously with PBS, VC-TK⁻, or VC-TK⁻-hFlt3L. FIG. 10Cconsists of two plots of the percentage of CD8⁺Granzyme B⁺ cells ininjected (right) and non-injected (left) tumors of mice treatedvariously with PBS, VC-TK⁻, or VC-TK⁻-hFlt3L. (*, p<0.05, ***, p<0.001).Data are means±SEM (n=3). FIG. 10D consists of two plots of thepercentage of CD4⁺Granzyme B⁺ cells in injected (right) and non-injected(left) tumors of mice treated variously with PBS, VC-TK⁻, orVC-TK⁻-hFlt3L. (**, p<0.01, ***, p<0.001). Data are means±SEM (n=3).

FIG. 11 is a gating strategy to separate CD11b⁺ DCs from CD103⁺ DCs inthe tumor infiltrating CD45⁺MHCII⁺ cells. Tumor-associated CD24⁺ DCs canbe further separated by their expression of CD11b and CD103. CD11b⁺ DCs(DC1) express a high level of CD11b, whereas CD103⁺ DCs (DC2) express ahigh level of CD103. F4/80⁺ tumor-associated macrophages can be furtherseparated into TAM1 and TAM2 based on their relative expression of CD11cand CD11b.

FIG. 12A-B is a series of graphical representations showing thatintratumoral injection of VC-TK⁻-hFlt3L leads to the modest increaseCD103⁺ DCs in the non-injected tumors. FIG. 12A is a graph showingpercentages of CD103⁺ DCs out of CD45+ cells in both injected andnon-injected tumors of mice treated with PBS, Heat-MVA, VC-TK−,VC-TK⁻-mGM-CSF, or VC-TK⁻hFlt3L (*, p<0.05). Data are means+/−SEM(n=3-4). FIG. 12B is a graph showing percentages of CD11b⁺ DCs out ofCD45+ cells in both injected and non-injected tumors (*, p<0.05). Dataare means+/−SEM (n=3-4).

FIG. 13A-E shows a series of graphical representations of intratumoralinjection of VC-TK or Heat-inactivated MVA in a 4T1 murine triplenegative breast carcinoma (TNBC) bilateral implantation model. 4T1 cells(2.5×10⁵) were implanted intradermally into the shaved skin on the rightflank, and (5×10⁴) cells were implanted to the left flank. At 5 dayspost implantation, the right side tumors (about 3 mm in diameter) wereinjected twice weekly with either PBS, VC-TK⁻ (2×10⁷ pfu), or with anequivalent amount of Heat-inactivated MVA. FIG. 13A-B are graphs of theinitial respective tumor volumes (injected and non-injected) prior tothe first injections. (FIG. 13 C, D are graphs of the respective tumorvolumes (injected and non-injected) at day 18 post the first injections.(*, P<0.05; ****, P<0.0001). (E) Kaplan-Meier survival curve of micetreated with PBS, VC-TK⁻, or Heat-iMVA. Survival data were analyzed bylog-rank (Mantel-Cox) test. (***, P<0.001).

FIG. 14A-E shows a series of graphical representations of intratumoraldelivery of VC-TK⁻-hFlt3L, Heat-inactivated MVA (Heat-iMVA) in anestablished large B16-F10 melanoma unilateral implantation model.B16-F10 cells (5×10⁵) were implanted intradermally to the right flank ofC57B/6 mice. At 9 days post implantation, when the average initial tumorvolumes reached 70 mm³, the tumors were injected with eitherVC-TK⁻-hFlt3L at 2×107 pfu or with an equivalent of Heat-iMVA twiceweekly. PBS was used as a control. (FIG. 14 A, B, C) are graphs ofvolume of injected tumors at various days post injection with PBS (A;n=10), or with Heat-iMVA (B, n=10), or with VC-TK⁻-hFlt3L (C; n=8). FIG.14D is a graph of initial tumor volumes of injected tumors at the timeof first injection. FIG. 14E is a Kaplan-Meier survival curve of micetreated with PBS, Heat-iMVA, or VC-TK⁻-hFlt3L. Survival data wereanalyzed by log-rank (Mantel-Cox) test. (***, P<0.001 for VC-TK⁻-hFlt3Lvs. PBS; ****, P<0.0001 for Heat-iMVA vs. PBS).

DETAILED DESCRIPTION Definitions

As used herein, the following terms shall have the meanings ascribed tothem below unless the context clearly indicates otherwise:

“Cancer” refers to a class of diseases of humans and animalscharacterized by uncontrolled cellular growth. Unless otherwiseexplicitly indicated, the term “cancer” may be used hereininterchangeably with the terms “tumor,” “malignancy,”“hyperproliferation” and “neoplasm(s);” the term “cancer cell(s)” isinterchangeable with the terms “tumor cell(s),” “malignant cell(s),”“hyperproliferative cell(s),” and “neoplastic cell(s)”.

“Melanoma” refers to a malignant neoplasm originating from cells thatare capable of producing melanin. The term melanoma is synonymous with“malignant melanoma”. Melanoma metastasizes widely, involving apatient's lymph nodes, skin, liver, lungs and brain tissues.

“Solid tumor” refers to all neoplastic cell growth and proliferation,and all pre-cancerous and cancerous cells and tissues, except forhematologic cancers such as lymphomas, leukemias and multiple myeloma.Examples of solid tumors include, but are not limited to: fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, andretinoblastoma. Some of the most common solid tumors for which thecompositions and methods of the present disclosure would be usefulinclude: head-and-neck cancer, rectal adenocarcinoma, glioma,medulloblastoma, urothelial carcinoma, pancreatic adenocarcinoma,endometrial cancer, ovarian cancer, prostate adenocarcinoma, non-smallcell lung cancer (squamous and adenocarcinoma), small cell lung cancer,melanoma, breast carcinoma, renal cell carcinoma, and hepatocellularcarcinoma.

“Metastasis” refers to the spread of cancer from its primary site toneighboring tissues or distal locations in the body. Cancer cells canbreak away from a primary tumor, penetrate into lymphatic and bloodvessels, circulate through the bloodstream, and grow in in normaltissues elsewhere in the body. Metastasis is a sequential process,contingent on tumor cells (or cancer stem cells) breaking off from theprimary tumor, traveling through the bloodstream or lymphatics, andstopping at a distant site. Once at another site, cancer cellsre-penetrate through the blood vessels or lymphatic walls, continue tomultiply, and eventually form a new tumor (metastatic tumor). In someembodiments, this new tumor is referred to as a metastatic (orsecondary) tumor.

“Immune response” refers to the action of one or more of lymphocytes,antigen presenting cells, phagocytic cells, granulocytes, and solublemacromolecules produced by the above cells or the liver (includingantibodies, cytokines, and complement) that results in selective damageto, destruction of, or elimination from the human body of cancerouscells, metastatic tumor cells, etc. An immune response may include acellular response, such as a T cell response that is an alteration(modulation, e.g., significant enhancement, stimulation, activation,impairment, or inhibition) of cellular function that is a T cellfunction. A T cell response may include generation, proliferation orexpansion, or stimulation of a particular type of T cell, or subset of Tcells, for example, CD4⁺ helper, CD8⁺ cytotoxic, or natural killer (NK)cells. Such T cell subsets may be identified by detecting one or morecell receptors or cell surface molecules (e.g., CD or cluster ofdifferentiation molecules). A T cell response may also include alteredexpression (statistically significant increase or decrease) of acellular factor, such as a soluble mediator (e.g., a cytokine,lymphokine, cytokine binding protein, or interleukin) that influencesthe differentiation or proliferation of other cells. For example, Type Iinterferon (IFN-α/β) is a critical regulator of the innate immunity(Huber et al. Immunology 132(4):466-474 (2011). Animal and human studieshave shown a role for IFN-α/β in directly influencing the fate of bothCD4⁺ and CD8⁺ T cells during the initial phases of antigen recognitionanti-tumor immune response. IFN Type I is induced in response toactivation of dendritic cells, in turn a sentinel of the innate immunesystem.

“Tumor immunity” refers to one or more processes by which tumors evaderecognition and clearance by the immune system. Thus, as a therapeuticconcept, tumor immunity is “treated” when such evasion is attenuated oreliminated, and the tumors are recognized and attacked by the immunesystem (the latter being termed herein “anti-tumor immunity”). Anexample of tumor recognition is tumor binding, and examples of tumorattack are tumor reduction (in number, size or both) and tumorclearance.

“T cell” refers to a thymus derived lymphocyte that participates in avariety of cell-mediated adaptive immune reactions.

“Helper T cell” refers to a CD4⁺ T cell; helper T cells recognizeantigen bound to MHC Class II molecules. There are at least two types ofhelper T cells, Th1 and Th2, which produce different cytokines.

“Cytotoxic T cell” refers to a T cell that usually bears CD8 molecularmarkers on its surface (CD8+) (but may also be CD4+) and that functionsin cell-mediated immunity by destroying a target cell having a specificantigenic molecule on its surface. Cytotoxic T cells also releaseGranzyme, a serine protease that can enter target cells via theperforin-formed pore and induce apoptosis (cell death). Granzyme servesas a marker of cytotoxic phenotype. Other names for cytotoxic T cellinclude CTL, cytolytic T cell, cytolytic T lymphocyte, killer T cell, orkiller T lymphocyte. Targets of cytotoxic T cells may includevirus-infected cells, cells infected with bacterial or protozoalparasites, or cancer cells. Most cytotoxic T cells have the protein CD8present on their cell surfaces. CD8 is attracted to portions of theClass I MHC molecule. Typically, a cytotoxic T cell is a CD8+ cell.

“Tumor-infiltrating lymphocytes” refers to white blood cells of asubject afflicted with a cancer (such as melanoma), that are resident inor otherwise have left the circulation (blood or lymphatic fluid) andhave migrated into a tumor.

“Immune checkpoint inhibitor(s)” or “immune checkpoint blocking agent”refers to molecules that completely or partially reduce, inhibit,interfere with or modulate the activity of one or more checkpointproteins. Checkpoint proteins regulate T-cell activation or function.Checkpoint proteins include, but are not limited to CTLA-4 and itsligands CD80 and CD86; PD-1 and its ligands PDL1 and PDL2; LAG3, B7-H3,B7-H4, TIM3, ICOS, and BTLA (Pardoll et al. Nature Reviews Cancer 12:252-264 (2012)).

“Parenteral” when used in the context of administration of a therapeuticsubstance includes any route of administration other than administrationthrough the alimentary tract. Particularly relevant for the methodsdisclosed herein are intravenous (including for example through thehepatic portal vein), intratumoral or intrathecal administration.

“Antibody” refers to an immunoglobulin molecule which specifically bindsto an antigen or to an antigen-binding fragment of such a molecule.Thus, antibodies can be intact immunoglobulins derived from naturalsources or from recombinant sources and can be immunoreactive(antigen-binding) fragments or portions of intact immunoglobulins. Theantibodies may exist in a variety of forms including, for example,polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, aswell as single chain antibodies (scFv) humanized antibodies, chimericantibodies, human recombinant antibodies and bi- and tri-specificantibodies.

“Oncolytic virus” refers to a virus that preferentially infects cancercells, replicates in such cells, and induces lysis of the cancer cellsthrough its replication process. Nonlimiting examples of naturallyoccurring oncolytic viruses include vesicular stomatitis virus,reovirus, as well as viruses engineered to be oncoselective such asadenovirus, Newcastle disease virus and herpes simplex virus (See, e.g.,Nemunaitis, J. Invest New Drugs. 17(4):375-86 (1999); Kirn, D H et al.Nat Rev Cancer. 9(1):64-71(2009); Kirn et al. Nat. Med. 7:781 (2001);Coffey et al. Science 282:1332 (1998)). Vaccinia virus infects manytypes of cells but replicates preferentially in tumor cells due to thefact that tumor cells (i) have a metabolism that favors replication,(ii) exhibit activation of certain pathways that also favor replicationand (iii) create an environment that evades the innate immune system,which also favors viral replication.

“Heat-inactivated” with particular reference to vaccinia viruses,including viral constructs harboring heterologous genes, such as GM-CSFand Flt3L, refers to a virus which has been further treated by exposureto heat under conditions that do not destroy its immunogenicity or itsability to enter target cells (tumor cells) but remove residualreplication ability of the virus as well as factors that inhibit thehost's immune response (for example, such factors as inhibit theinduction of IFN Type I in infected cells). An example of suchconditions is exposure to a temperature within the range of about 50 toabout 60° C. for a period of time of about an hour. Other times andtemperatures can be determined with routine experimentation and IFN TypeI induction in infected cDC's can be compared to the Heat-inactivatedvirus used in experiments described herein and should be higher thanthat of vaccinia virus.

“UV-inactivated” with particular reference to vaccinia viruses,including viral constructs harboring heterologous genes, such as GM-CSFand Flt3L, refers to a virus which has been inactivated by exposure toUV under conditions that do not destroy its immunogenicity or itsability to enter target cells (tumor cells) but remove residualreplication ability of the virus. An example of such conditions, whichcan be useful in the present methods, is exposure to UV using forexample a 365 nm UV bulb for a period of about 30 min to about 1 hour(Tsung et al. J Virol 70,165-171 (1996); Drillien, R. et al. J Gen Virol85: 2167-2175 (2004)).

“Subject” means any animal (mammalian, human or other) patient that canbe afflicted with cancer.

“Therapeutically effective amount” or “effective amount” refers to asufficient amount of an agent when administered at one or more dosagesand for a period of time sufficient to provide a desired biologicalresult in alleviating, curing or palliating a disease. In the presentdisclosure, an effective amount of the E3LΔ83N-TK⁻, E3LΔ83N-TK⁻-mGM-CSF,and E3LΔ83N-TK⁻-hFlt3L and corresponding inactivated viruses is anamount that (administered for a suitable period of time and at asuitable frequency) accomplishes one or more of the following: reducesthe number of cancer cells; or reduces the tumor size or eradicates thetumor; inhibits (i.e., slows down or stops) cancer cell infiltrationinto peripheral organs; inhibits (i.e., slows down or stops) metastaticgrowth; inhibits (i.e., stabilizes or arrests) tumor growth; allows fortreatment of the tumor, and induces an immune response against thetumor. An appropriate therapeutic amount in any individual case may bedetermined by one of ordinary skill in the art using routineexperimentation in light of the present disclosure. Such determinationwill begin with amounts found effective in vitro and amounts foundeffective in animals. The therapeutically effective amount will beinitially determined based on the concentration or concentrations foundto confer a benefit to cells in culture. Effective amounts can beextrapolated from data within the cell culture and can be adjusted up ordown based on factors such as detailed herein. An example of aneffective amount range is from 10⁵ viral particles to about 10¹² viralparticles per administration.

With particular reference to the viral-based immunostimulatory agentsdisclosed herein, “therapeutically effective amount” or “effectiveamount” refers to an amount of a composition comprising E3LΔ83N-TK⁻,E3LΔ83N-TK⁻-GM-CSF, E3LΔ83N-TK⁻-hFlt3L and/or a correspondinginactivated virus sufficient to reduce, inhibit, or abrogate tumor cellgrowth, thereby reducing or eliminating the tumor, or sufficient toinhibit, reduce or abrogate metastatic spread either in vitro or in asubject or to elicit an immune response against the tumor that willeventually result in one or more of reduction, inhibition and/orabrogation as the case may be. The reduction, inhibition, or eradicationof tumor cell growth may be the result of necrosis, apoptosis, or animmune response or a combination of two or more of the foregoing. Theamount that is therapeutically effective may vary depending on suchfactors as the particular E3LΔ83N-TK⁻,E3LΔ83N-TK⁻-GM-CSF,E3LΔ83N-TK⁻-hFlt3L and/or corresponding inactivatedviruses used in the composition, the age and condition of the subjectbeing treated, the extent of tumor formation, the presence or absence ofother therapeutic modalities, and the like. Similarly, the dosage of thecomposition to be administered and the frequency of its administrationwill depend on a variety of factors, such as the potency of the activeingredient, the duration of its activity once administered, the route ofadministration, the size, age, sex and physical condition of thesubject, the risk of adverse reactions and the judgment of the medicalpractitioner. The compositions are administered in a variety of dosageforms, such as injectable solutions.

With particular reference to combination therapy with an immunecheckpoint inhibitor, “therapeutically effective amount” for an “immunecheckpoint blocking or blockade agent” shall mean an amount of an immunecheckpoint blocking agent sufficient to block an immune checkpoint fromaverting apoptosis response in tumor cells of the subject being treated.There are several immune checkpoint blocking agents approved, inclinical trials or still otherwise under development including CD28inhibitors such as CTL4 inhibitors (e.g., ipilimumab), PD-1 inhibitors(e.g., nivolumab, pembrolizumab, pidilizumab, lambrolizumab), II DLBCLinhibitors such as AMP-224, PD-L1 inhibitors (MPDL3280A, BMS-936559,MEDI4736, MSB 00107180) ICOS and BTLA or decoy molecules of them. Dosageranges of the foregoing are known in or readily within the skill in theart as several dosing clinical trials have been completed, makingextrapolation to other agents possible.

Preferably, the tumor expresses the particular checkpoint. While this isdesirable, it is not strictly necessary as immune checkpoint blockingagents block more generally immune suppressive mechanisms within thetumors, elicited by tumor cells, stromal cell, and tumor infiltratingimmune cells.

For example, the CTLA4 inhibitor ipilimumab, when administered asadjuvant therapy after surgery in melanoma is administered at 1-2 mg/mLover 90 minutes for a total infusion amount of 3 mg/kg every three weeksfor a total of 4 doses. This therapy is often accompanied by severe evenlife-threatening immune-mediated adverse reactions, which limits thetolerated dose as well as the cumulative amount that can beadministered. This and other checkpoint blockade inhibitors,administered alone, are commonly given in amounts per dose rangingbetween 1 and 3 mg/mL(as shown below). It is anticipated that it will bepossible to reduce the dose and/or cumulative amount of ipilimumab whenit is administered conjointly with one or more of E3LΔ83N-TK⁻,E3LΔ83N-TK⁻-GM-CSF, and E3LΔ83N-TK⁻-hFlt3L viruses. In particular, inlight of the experimental results set forth below, it is anticipatedthat it will be further possible to reduce the CTLA4 inhibitor's dose ifit is administered directly to the tumor simultaneously or sequentiallywith one or more of E3LΔ83N-TK⁻, E3LΔ83N-TK⁻-GM-CSF, andE3LΔ83N-TK⁻-hFlt3L viruses and corresponding inactivated viralconstructs. Accordingly, the amounts provided above for ipilimumab willbe a starting point for determining the particular dosage and cumulativeamount to be given to a patient in conjoint administration but dosingstudies will be required to determine optimum amounts.

Pembrolizumab is prescribed for administration as adjuvant therapy inmelanoma diluted to 25 mg/mL is administered at a dosage of 2 mg/kg over30 minutes every three weeks.

Nivolumab is prescribed for administration at 3 mg/kg as an intravenousinfusion over 60 minutes every two weeks. For therapeuticuses/applications human GM-CSF will be utilized. In the Examplesdescribed herein, mouse GM-CSF described as mGM-CSF is used in themodel/experimental systems. Additionally, it is expected that multipletreatments with any one or more of the viruses of the present disclosurecan be administered in multiple doses until the tumors resolve or are nolonger responding to the treatment.

It will be understood that the foregoing combination therapies of one ormore viruses with a checkpoint blockade inhibitor can be administered byone or more practitioners acting under each other's instructions oroperating as a team.

“Pharmaceutically acceptable carrier and/or diluent” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for biologically active substances is well knownin the art. Supplementary active ingredients, such as antimicrobials,can also be incorporated into the compositions.

“Delivering” used in connection with depositing one or more ofE3LΔ83N-TK⁻, E3LΔ83N-TK⁻-GM-CSF, and E3LΔ83N-TK⁻-Flt3L viruses of thepresent disclosure in the tumor microenvironment whether this is done bylocal administration to the tumor or by for example intravenous route.The term focuses on E3LΔ83N-TK⁻, E3LΔ83N-TK⁻-GM-CSF, E3LΔ83N-TK⁻-hFlt3Lviruses that reaches the tumor itself. “Delivering” is synonymous withadministering but it is used with a particular administration locale inmind e.g. intratumoral.

In the present disclosure, the inventors generated recombinantE3LΔ83N-TK⁻ virus expressing human Flt3L, with the goal of deliveringthis growth factor to the tumor microenvironment to facilitaterecruitment, differentiation and function of immune cells, includingCD103⁺/CD8α dendritic cells (DCs). A somewhat similar strategy has beenused and proven to be effective in the clinical development of JX-594 byJennerex, in which vaccinia virus is engineered to express a transgeneencoding granulocyte-macrophage colony stimulating factor (GM-CSF) withthe deletion of vaccinia thymidine kinase (TK) gene to increase tumorselectivity. GM-CSF is another important growth factor for DChomeostasis at the peripheral non-lymphoid tissues (King et al., 2010;Greter et al., 2012). Melanoma vaccine (GVAX) comprises lethallyirradiated allogeneic melanoma cells secreting GM-CSF has shown someclinical benefit (Dranoff et al., 2003). Curran and Allison showed thatthe combination of B16-GMCSF (GVAX) or B16-Flt3L (F13 VAX) with CTLA-4blocking agent eradicated established melanoma in about 60% of the miceif the vaccines were administered at distal sites from the tumors(Curran and Allison, 2009). However, when the vaccines were administeredto the tumors in combination with CTLA-4 blocking agent, GVAX wasineffective in tumor eradication, whereas Fl3 VAX treatment resulted in75% of tumor-free mice. One potential explanation is that GM-CSFadministration to the tumors might induce myeloid suppressor cellgeneration within the tumor (Serafini et al., 2004). With the concernthat administration of GM-CSF to the tumors might induce immunetolerance, inventors of the present disclosure performed head-to-headcomparisons of two recombinant viruses, with VC-TK⁻ as a vectorexpressing hFlt3L or GM-CSF, and vector alone, for eradication ofestablished B16 melanoma. The inventors discovered that VC-TK⁻-hFlt3L ismore efficacious than VC-TK⁻-mGM-CSF or vector alone in eradicating orcontrolling tumor growth (Example 6). As described in Example 6, theinventors showed that while intratumor injection of attenuatedreplication competent VC-TK⁻, VC-TK⁻-mGM-CSF, or VC-TK⁻-hFlt3L caneffectively eradicate injected tumors, intratumoral delivery ofVC-TK⁻-hFlt3L is more efficacious than VC-TK⁻-mGM-CSF in delaying thegrowth of contralateral tumor and extending survival. This systemiceffect of VC-TK⁻-hFlt3L is important not only for the treatment ofnoninjected tumors, but also for the treatment of metastatic disease.

Additionally, the inventors of the present disclosure have shown thatintratumoral delivery of oncolytic viruses overcomes the resistance toimmune checkpoint blocking agents. As shown in Example 7, thecombination of intratumoral delivery of either VC-TK⁻-mGM-CSF, orVC-TK⁻-hFlt3L with systemic delivery of anti-CTLA-4 antibody lead to theeradication of 10/10 injected tumors, and significant delay of thegrowth of contralateral non-injected tumors, as well as completeeradication of tumors in 40-60% of the cases. However, intratumoraldelivery of VC-TK⁻-hFlt3L in combination with anti-CTLA-4 antibody wasmore efficacious than VC-TK⁻-mGM-CSF in combination with anti-CTLA-4antibody in delaying the growth of contralateral tumor and extendingsurvival of treated mice. These results indicate for the first time thatVC-TK⁻-hFlt3L may provide a successful and indeed a superior option forthe treatment of melanoma patients, alone or in combination with immunecheckpoint blocking agents.

In the present disclosure, the inventors further explored whetherinactivated VC-TK⁻-mGM-CSF strain can be used as cancerimmunotherapeutic agent. In fact, they observed that intratumoraldelivery of Heat-inactivated VC-TK⁻-mGM-CSF is more efficacious ineradiating tumors and generating antitumoral adaptive immunity than liveVC-TK⁻-mGM-CSF (Example 8). Thus, as a treatment option, patients can betreated with Heat-inactivated VC-TK⁻-mGM-CSF in order to achieveimproved treatment results. It is anticipated that similar results willbe observed if instead of heat-inactivating the virus, ultravioletirradiation (UV) inactivation is employed instead.

Furthermore, the inventors of the present disclosure have shown that thecombination of intratumoral injection of VC-TK⁻-mGM-CSF orHeat-inactivated VC-TK⁻-mGM-CSF with intraperitoneal delivery of immunecheckpoint blocking agent leads to synergistic therapeutic effects(Example 9).

In one embodiment, the present disclosure relates to a method foreliciting an antitumor immune response in subjects with tumorscomprising delivering to the tumor an effective amount of one or more ofE3LΔ83N-TK⁻, E3LΔ83N-TK⁻-GM-CSF, and E3LΔ83N-TK⁻-hFlt3L viruses.Stimulation of the immune system may be manifest by one or more of thefollowing immunological effects:

an increase in cytotoxic CD8+ T cells within the tumor and/or intumor-draining lymph nodes;

induction of maturation of dendritic cells infiltrating said tumorthrough induction of type I IFN;

induction of activated T helper cells in the subject recognizing tumorcells within the tumor and/or in tumor draining lymph nodes

increase of CD103⁺ dendritic cells in noninjected tumors of the subject(especially for the hFLT3L construct).

The foregoing one or more immunological effects may serve as earlyindicators of response of the subject to the treatment and may serve asmonitors of the continued effectiveness of same.

In one embodiment, the present disclosure provides a method of treatinga subject diagnosed with a solid tumor comprising delivering to thetumor a therapeutic effective amount of one or more of E3LΔ83N-TK⁻,E3LΔ83N-TK⁻-GM-CSF, and E3LΔ83N-TK⁻-hFlt3L viruses.

In one embodiment, the present disclosure provides a method for inducinganti-tumor immunity in a subject diagnosed with cancer comprisingadministering to the subject a therapeutically effective amount of oneor more of E3LΔ83N-TK⁻, E3LΔ83N-TK⁻-GM-CSF, and E3LΔ83N-TK⁻-hFlt3Lviruses. The methods of the present disclosure include induction ofanti-tumor immunity that can reduce the size of the tumor, eradicate thetumor, inhibit growth of the tumor, inhibit metastasis or metastaticgrowth of the tumor, induce apoptosis of tumor cells or prolong survivalof the subject (compared to untreated or conventionally treatedsubjects).

In another embodiment, the present disclosure provides a method forenhancing, stimulating, or eliciting, in a subject diagnosed with asolid malignant tumor, an anti-tumor immune response that may include aninnate immune response and/or an adaptive immune response such as a Tcell response by exposing the tumor to one or more of E3LΔ83N-TK⁻,E3LΔ83N-TK⁻-GM-CSF, and E3LΔ83N-TK⁻-hFlt3L viruses in a therapeuticallyeffective amount.

In specific embodiments, the present disclosure provides methods ofeliciting an immune response that mediates adaptive immune responsesboth in terms of T-cell cytotoxicity directed against tumor cells and interms of eliciting T helper cells also directed against tumor cells. Themethods comprise administering to a subject afflicted with a solid tumorintratumorally or intravenously a composition comprising one or more ofE3LΔ83N-TK⁻, E3LΔ83N-TK⁻-GM-CSF, and E3LΔ83N-TK⁻-hFlt3L viruses whereinadministration of said composition results in a tumor-specific immuneresponse against the tumor and, eventually, in reduction, inhibition oreradication of tumor growth, in inhibition of metastatic growth,apoptosis of tumor cells and/or prolongation of the subject's survival.Indeed the present inventors have shown that cancer cells are beingkilled and that the immune response can migrate to remote locations, aswould be the case with metastases.

In some embodiments, the present disclosure provides methods ofeliciting an immune response that mediates adaptive immune responsesboth in terms of T-cell cytotoxicity directed against tumor cells and interms of eliciting T helper cells also directed against tumor cells. Themethods comprise administering to a subject parenterally a compositioncomprising one or more of E3LΔ83N-TK⁻, E3LΔ83N-TK⁻-mGM-CSF, andE3LΔ83N-TK⁻-hFlt3L viruses wherein administration of said compositionresults in a tumor-specific immune response against the tumor and,eventually, in reduction, inhibition or eradication of tumor growthand/or in inhibition of metastatic growth, apoptosis of tumor cellsand/or prolongation of survival of the treated subject. Forintraperitoneal metastases, the viruses can be injectedintraperitoneally. For brain metastasis, the viruses can be injectedintratumorally under stereotactic guidance, or intrathecally.

Indeed the present inventors have shown that cancer cells are beingkilled and that the immune response can migrate to remote locations, aswould be the case with metastases.

The present disclosure thus provides a method for treating a solidmalignant tumor, delivering to a tumor of the subject an amount ofE3LΔ83N-TK⁻, E3LΔ83N-TK⁻-GM-CSF, and/or E3LΔ83N-TK⁻-hFlt3L viruseffective to induce a therapeutic immune response in a subject diagnosedwith solid tumor.

As is shown herein, current literature, and without wishing to be boundby theory, the following mechanisms are believed to contribute toanti-tumor effects of E3LΔ83N-TK⁻, E3LΔ83N-TK⁻-GM-CSF, andE3LΔ83N-TK⁻-hFlt3L viruses: (i) oncolysis of tumor cells and release oftumor antigens; (ii) induction of cytotoxic CD8+ and effector CD4⁺ Tcells in the tumors and tumor draining lymph nodes; (iii) alteration oftumor immune suppressive environment through the release of viral DNAand RNA; and (iv) induction of anti-tumor antibodies.

Immune Response

In addition to induction of the immune response by up-regulation ofparticular immune system activities (such as antibody and/or cytokineproduction, or activation of cell mediated immunity), immune responsesmay also include suppression, attenuation, or any other down-regulationof detectable immunity, so as to reestablish homeostasis and preventexcessive damage to the host's own organs and tissues. In someembodiments, an immune response that is induced according to the methodsof the present disclosure generates cytotoxic CD8⁺ T cells or activatedT helper cells or both that can bring about directly or indirectly thedeath, or loss of the ability to propagate, of a tumor cell.

Induction of an immune response by the methods of the present disclosuremay be determined by detecting any of a variety of well-knownimmunological parameters (Takaoka et al., Cancer Sci. 94:405-11 (2003);Nagorsen et al., Crit. Rev. Immunol. 22:449-62 (2002)). Induction of animmune response may therefore be established by any of a number ofwell-known assays, including immunological assays, Such assays include,but need not be limited to, in vivo, ex vivo, or in vitro determinationof soluble immunoglobulins or antibodies; soluble mediators such ascytokines, chemokines, hormones, growth factors and the like as well asother soluble small peptide, carbohydrate, nucleotide and/or lipidmediators; cellular activation state changes as determined by alteredfunctional or structural properties of cells of the immune system, forexample cell proliferation, altered motility, altered intracellularcation gradient or concentration (such as calcium); phosphorylation ordephosphorylation of cellular polypeptides; induction of specializedactivities such as specific gene expression or cytolytic behavior;cellular differentiation by cells of the immune system, includingaltered surface antigen expression profiles, or the onset of apoptosis(programmed cell death); or any other criterion by which the presence ofan immune response may be detected. For example, cell surface markersthat distinguish immune cell types may be detected by specificantibodies that bind to CD4⁺, CD8⁺, or NK cells. Other markers andcellular components that can be detected include but are not limited tointerferon γ (IFN-γ), tumor necrosis factor (TNF), IFN-α, IFN-β, IL-6,and CCL5. Common methods for detecting the immune response include, butare not limited to flow cytometry, ELISA, immunohistochemistry.Procedures for performing these and similar assays are widely known andmay be found, for example in Letkovits (Immunology Methods Manual: TheComprehensive Sourcebook of Techniques, Current Protocols in Immunology,1998).

Pharmaceutical Compositions and Preparations

Pharmaceutical compositions comprising E3LΔ83N-TK⁻, E3LΔ83N-TK⁻-GM-CSF,and/or E3LΔ83N-TK⁻-hFlt3L viruses may contain a carrier or diluent,which can be a solvent or dispersion medium containing, for example,water, polyol (for example, glycerol, propylene glycol, and liquidpolyethylene glycol, and the like), suitable mixtures thereof, andvegetable oils. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can beeffected by various antibacterial and antifungal agents, for example,parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.In many cases, it will be preferable to include isotonic agents, forexample, sugars or sodium chloride. Prolonged absorption of theinjectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Pharmaceutical compositions and preparations comprising E3LΔ83N-TK⁻,E3LΔ83N-TK⁻-GM-CSF, and/or E3LΔ83N-TK⁻-hFlt3L viruses may bemanufactured by means of conventional mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping orlyophilizing processes. Pharmaceutical viral compositions may beformulated in conventional manner using one or more physiologicallyacceptable carriers, diluents, excipients or auxiliaries that facilitateformulating virus preparations suitable for in vitro, in vivo, or exvivo use. The compositions can be combined with one or more additionalbiologically active agents (for example parallel administration ofGM-CSF) and may be formulated with a pharmaceutically acceptablecarrier, diluent or excipient to generate pharmaceutical (includingbiologic) or veterinary compositions of the instant disclosure suitablefor parenteral or intra-tumoral administration. Suitable excipientvehicles include, for example, water, saline, dextrose, glycerol,ethanol, inert proteins, hydrophillic polymers, amino acids, fattyacids, surfactants, non-ionic surfactants, carbohydrates, dextrins,polyols, chelating agents, or the like, and combinations thereof. Inaddition, if desired, the vehicle may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents or pH buffering agents.Actual methods of preparing such dosage forms are known, or will beapparent, to those skilled in the art. See, e.g., Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 17thedition, 1985; Remington: The Science and Practice of Pharmacy, A. R.Gennaro, (2000) Lippincott, Williams & Wilkins.

Many types of formulation are possible and well-known. The particulartype chosen is dependent upon the route of administration chosen, as iswell-recognized in the art. For example, systemic formulations willgenerally be designed for administration by injection, e.g.,intravenous, as well as those designed for intratumoral delivery.Preferably, the systemic or intratumoral formulation is sterile.

Sterile injectable solutions are prepared by incorporating E3LΔ83N-TK⁻,E3LΔ83N-TK⁻-GM-CSF, and/or E3LΔ83N-TK⁻-hFlt3L viruses in the requiredamount of the appropriate solvent with various other ingredientsenumerated herein, as required, followed by suitable sterilizationmeans. Generally, dispersions are prepared by incorporating the varioussterilized active ingredients into a sterile vehicle that contains thebasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying techniques, which yield a powder of theinactive-E3LΔ83N-TK⁻, E3LΔ83N-TK⁻-GM-CSF, and/or E3LΔ83N-TK⁻-hFlt3Lviruses, and plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Dosage of E3LΔ83N-TK⁻, E3LΔ83N-TK⁻-GM-CSF, E3LΔ83N-TK⁻-hFlt3L Viruses

In general, the subject is administered a unit dosage of E3LΔ83N-TK⁻,E3LΔ83N-TK⁻-GM-CSF, and/or E3LΔ83N-TK⁻-hFlt3L viruses in the range ofabout 10⁵ to about 10¹⁰ plaque forming units (pfu), although a lower orhigher dose may be administered. In a preferred embodiment, dosage isabout 10⁶-10⁹ pfu. Typically, a unit dosage is administered in a volumewithin the range from 1 to 10 ml. The equivalence of pfu to virusparticles can differ according to the specific pfu titration methodused. Generally, pfu is equal to about 5 to 100 virus particles. Atherapeutically effective amount of E3LΔ83N-TK⁻, E3LΔ83N-TK⁻-GM-CSF,E3LΔ83N-TK⁻-hFlt3L viruses can be administered in one or more divideddoses for a prescribed period of time and at a prescribed frequency ofadministration. For example, therapeutically effective amount ofE3LΔ83N-TK⁻, E3LΔ83N-TK⁻-GM-CSF, E3LΔ83N-TK⁻-hFlt3L viruses inaccordance with the present disclosure may vary according to factorssuch as the disease state, age, sex, weight, and general condition ofthe subject, and the ability of E3LΔ83N-TK⁻, E3LΔ83N-TK⁻-GM-CSF,E3LΔ83N-TK⁻-hFlt3L viruses to elicit a desired immunological response inthe particular subject.

As is apparent to persons working in the field of cancer therapy,variation in dosage will necessarily occur depending for example on thecondition of the subject being treated, route of administration and thesubject's response to the therapy. In delivering E3LΔ83N-TK⁻,E3LΔ83N-TK⁻-GM-CSF, and/or E3LΔ83N-TK⁻-hFlt3L viruses to a subject, thedosage will also vary depending upon such factors as the general medicalcondition, previous medical history, disease progression, tumor burden,ability to mount an immune response, and the like.

It may be advantageous to formulate compositions of present disclosurein dosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the mammalian subjects to be treated; eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect in association with therequired pharmaceutically or veterinary acceptable carrier.

Administration and Therapeutic Regimen of E3LΔ83N-TK⁻,E3LΔ83N-TK⁻-GM-CSF, and/or E3LΔ83N-TK⁻-hFlt3L viruses

Administration of one or more of E3LΔ83N-TK⁻, E3LΔ83N-TK⁻-GM-CSF,E3LΔ83N-TK⁻-hFlt3L viruses can be achieved using a combination ofroutes, including parenteral, intratumoral, intrathecal or intravenousadministration. In one embodiment, one or more of E3LΔ83N-TK⁻,E3LΔ83N-TK⁻-GM-CSF, E3LΔ83N-TK⁻-hFlt3L viruses are administered directlyinto the tumor, e.g. by intratumoral injection, where a direct localreaction is desired. Additionally, administration routes of E3LΔ83N-TK⁻,E3LΔ83N-TK⁻-GM-CSF, and/or E3L483N-TK⁻-hFlt3L viruses can vary, e.g.,first administration using an intratumoral injection, and subsequentadministration via an intravenous injection, or any combination thereof.A therapeutically effective amount of one or more of E3LΔ83N-TK⁻,E3LΔ83N-TK⁻-GM-CSF, and E3LΔ83N-TK⁻-hFlt3L viruses injection can beadministered for a prescribed period of time and at a prescribedfrequency of administration. In certain embodiments, E3LΔ83N-TK⁻,E3LΔ83N-TK⁻-GM-CSF, E3LΔ83N-TK⁻-hFlt3L viruses can be used inconjunction with other therapeutic treatments. For example, E3LΔ83N-TK⁻,E3LΔ83N-TK⁻-GM-CSF, and/or E3LΔ83N-TK⁻-hFlt3L viruses can beadministered in a neoadjuvant (preoperative) or adjuvant (postoperative)setting for subjects inflicted with bulky primary tumors. It isanticipated that such optimized therapeutic regimen will induce animmune response against the tumor, and reduce the tumor burden in asubject before or after primary therapy, such as surgery. Furthermore,E3LΔ83N-TK⁻, E3LΔ83N-TK⁻-GM-CSF, and E3LΔ83N-TK⁻-hFlt3L viruses can beadministered in conjunction with other therapeutic treatments such aschemotherapy or radiation.

In certain embodiments, the E3LΔ83N-TK⁻, E3LΔ83N-TK⁻-GM-CSF, andE3LΔ83N-TK⁻-hFlt3L viruses are administered at least once weekly ormonthly but can be administered more often if needed, such as two timesweekly for several weeks, months, years or even indefinitely as long asbenefit persist. More frequent administrations are contemplated iftolerated and if they result in sustained or increased benefits.Benefits of the present methods include but are not limited to thefollowing: reduction of the number of cancer cells, reduction of thetumor size, eradication of tumor, inhibition of cancer cell infiltrationinto peripheral organs, inhibition or stabilization of metastaticgrowth, inhibition or stabilization of tumor growth, and stabilizationor improvement of quality of life. Furthermore, the benefits may includeinduction of an immune response against the tumor, activation of Thelper cells, an increase of cytotoxic CD8⁺ T cells, or reduction ofregulatory CD4⁺ cells. For example, in the context of melanoma or, abenefit may be a lack of recurrences or metastasis within one, two,three, four, five or more years of the initial diagnosis of melanoma.Similar assessments can be made for colon cancer and other solid tumors.

In certain other embodiments, the tumor mass or tumor cells are treatedwith one or more of E3LΔ83N-TK⁻, E3LΔ83N-TK⁻-GM-CSF, andE3LΔ83N-TK⁻-hFlt3L viruses in vivo, ex vivo, or in vitro.

Kits

The present disclosure contemplates the provision of kits comprising oneor more compositions comprising one or more of the E3LΔ83N-TK⁻,E3LΔ83N-TK⁻-GM-CSF, or E3LΔ83N-TK⁻-hFlt3L viruses described herein. Thekit can comprise one or multiple containers or vials of the virus,together with instructions for the administration of the virus to asubject to be treated. The instructions may indicate a dosage regimenfor administering the composition or compositions as provided below.

In some embodiments, the kit may also comprise an additional compositioncomprising a checkpoint inhibitor for conjoint administration with anyof the virus compositions described herein.

EXAMPLES Materials and Methods

Viruses and Cell Lines

E3LΔ83N (VC) and ΔF3L (VI) viruses were kindly provided by B. L. Jacobs(Arizona State University). They were propagated in BSC40 cells andviral titers were determined by plaque assay using BSC40 cells. VC-TK⁻,VC-TK⁻-mGM-CSF, VC-TK⁻-hFlt3L viruses were generated through homologousrecombination at the thymidine kinase (TK) locus (see Example 2). Theserecombinant viruses were enriched through culturing in gpt selectionmedium and plaque purified in the presence of selection medium throughmore than five rounds. The pure recombinant clones were amplified in theabsence of selection medium. After validation, the viruses were purifiedthrough a 36% sucrose cushion.

MVA virus was kindly provided by Gerd Sutter (University of Munich),propagated in BHK-21 (baby hamster kidney cell, ATCC CCL-10) cells.Heat-inactivated MVA and Heat-inactivated VC-TK⁻-mGM-CSF were generatedby incubating purified respective viruses at 55° C. for 1 hour.Heat-inactivation led to reduction of infectivity by 1,000-fold.

BSC40 cells were cultured in Dulbecco's modified Eagle's medium (DMEM)supplemented containing 10% FBS, 0.1 mM nonessential amino acids (NEAA),and 50 mg/ml gentamycin. RK13 (rabbit kidney) cells were cultured inmodified Eagle's medium containing 10% FBS, 0.1 mM nonessential aminoacids, and 50 g/ml gentamicin. The murine melanoma cell line B16-F10 wasoriginally obtained from I. Fidler (MD Anderson Cancer Center). B16-F10cells were maintained in RPMI 1640 medium supplemented with 10% fetalbovine serum (FBS), 100 Units/ml penicillin, 100 μg/ml streptomycin, 0.1mM NEAA, 2 mM L-glutamine, 1 mM sodium pyruvate, and 10 mM HEPES buffer.The human melanoma SK-MEL-39, SK-MEL-188, SK-MEL90, SK-MEL90, andSK-MEL-28 cells were cultured in MEM medium supplemented with 10% FBSand 4 mm L-Glutamine. All cells were grown at 37° C. in a 5% CO₂incubator.

Murine triple negative breast cancer cell line 4T1 was cultured in theRPMI medium with 10% FBS.

One-Step Growth in Cell Culture

B16-F10 cells and human melanoma cells were cultured overnight prior toinfection with viruses, including ΔE3L (VI), E3LΔ83N (VC), VC-TK⁻,VC-TK⁻-mGM-CSF, VC-TK⁻-hFlt3L) at a low MOI. The inoculum was removedafter 60 min; the cells were washed twice with PBS and then overlaidwith medium. The cells were harvested at 1, 4, 12, 24, 48, and somecases 72 h after initial infection by scraping the cells into 1 ml ofmedium. After three cycles of freezing and thawing, the samples weresonicated and virus titers (for all of the viruses except for ΔE3L) weredetermined by serial dilution and infection of BSC40 cell monolayers.ΔE3L viral titers were determined on RK13 cells. Plaques were visualizedby staining with 0.1% crystal violet in 20% ethanol.

Western Blot Analysis

Murine melanoma B16-F10 cells or human melanoma cells SK-MEL-28,SK-MEL146 (1×10⁶) were infected with E3LΔ83N-TK⁻-mGM-CSF orE3LΔ83N-TK⁻-hFlt3L viruses at a MOI (multiplicity of infection) of 10.At various times post-infection, the supernatants and cell lysates werecollected. Equal amounts of proteins were subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis and the polypeptides weretransferred to a nitrocellulose membrane. The level of mGM-CSF andhFlt3L expression was determined by using an anti-mGM-CSF or anti-hFlt3Lantibody. Anti-glyceraldehyde-3-phosphate dehydrogenase (GADPH) antibody(Cell Signaling) was used as a loading control.

Mice

Female C57BL/6J and BALB/c mice between 6 and 8 weeks of age werepurchased from the Jackson Laboratory and were used for in vivo tumorimplantation and treatment experiments. These mice were maintained inthe animal facility at the Sloan Kettering Institute. All procedureswere performed in strict accordance with the recommendations in theGuide for the Care and Use of Laboratory Animals of the NationalInstitute of Health. The protocol was approved by the Committee on theEthics of Animal Experiments of Sloan Kettering Cancer Institute.

Tumor Implantation and Intratumoral Injection with Viruses in thePresence or Absence of Systemic or Intratumoral Administration of ImmuneCheckpoint Blockade

B16-F10 melanoma cells (5×10⁵) were implanted intradermally into theshaved skin on the right flank a C57BL/6J mouse, whereas fewer cells(1×10⁵) were implanted to the left flank of the same mouse. After 7 to 8days post implantation, tumor sizes were measured and tumors that are 3mm in diameter or larger on the right flank of the mice were injectedwith VC-TK⁻, VC-TK⁻-mGM-CSF, VC-TK⁻-hFlt3L viruses (2×10⁷ pfu) or PBSwhen the mice were under anesthesia. Viruses were injected twice weekly.Mice were monitored daily and tumor sizes were measured twice a week.Tumor volumes were calculated according the following formula: 1(length)×w (width)×h(height)/2. The survival of mice was monitored. Micewere euthanized for signs of distress or when the diameter of the tumorreached 10 mm. In some experimental groups, the mice were treated withintraperitoneal delivery of anti-CTLA-4 (100 μg per mouse) or anti-PD-L1antibodies (250 μg per mouse) twice weekly.

In some experiments, 4T1 murine triple negative breast cancer (TNBC)cells were implanted intradermally to the left and right flanks ofBALB/c mice (2.5×10⁵ to the right flank and 5×10⁴ to the left flank). 5days post tumor implantation, the larger tumors on the right flank wereinjected with either Heat-iMVA or VC-TK⁻ virus (2×10⁷ pfu) twice weekly.Mice were monitored daily and tumor sizes were measured twice a week.The survival of mice was monitored.

Unilateral Intradermal Tumor Implantation and Intratumoral Injectionwith Viruses

B16-F10 melanoma (5×10⁵ cells in a volume of 50 μl) were implantedintradermally into the shaved skin on the right flank of WT C57BL/6Jmice. After 9 days post implantation, tumor sizes were measured andtumors that are 5-6 mm in diameter were injected with Heat-iMVA(equivalent of 2×10⁷ pfu of MVA in a volume of 50 μl) or withVC-TK⁻-mGM-CSF, or with PBS when the mice were under anesthesia twiceweekly. Mice were monitored daily and tumor sizes were measured twice aweek. Tumor volumes were calculated according the following formula:1(length)×w (width)×h(height)/2. Mice were euthanized for signs ofdistress or when the diameter of the tumor reached 15 mm.

Tumor Challenge to Assess the Development of Cross-Protective AntitumorImmunity

The surviving mice (more than 60 days post initiation of intratumoralvirotherapy) and nave mice were challenged with intradermally deliveryof a lethal dose of MC38 (1×10⁵ cells) to assess cross-protectiveimmunity against heterologous tumors.

Preparation of Tumor Cell Suspensions

To analyze immune cell phenotypes and characteristics in the tumors, wegenerated cell suspensions prior to FACS analysis according to thefollowing protocol (Zamarin et al., Science Translational Medicine 6,226-232 (2014)). First we isolated injected and/or non-injected tumorsusing forceps and surgical scissors three days post second treatment and7 days post first treatment with PBS or viruses. The tumors were thenweighed. Tumors were minced prior to incubation with Liberase (1.67Wünsch U/ml) and DNase (0.2 mg/ml) for 30 minutes at 37° C. Cellsuspensions were generated by repeated pipetting, filtered through a70-μm nylon filter, and then washed with complete RPMI.

Flow Cytometry Analysis of Tumor Infiltrating Immune Cells

In the bilateral tumor implantation model, 5×10⁵ B16-F10 melanoma cellswere implanted intradermally to the right flank and 2.5×10⁵ cells to theleft flank of C57B/6 mice. Seven days post implantation, either VC-TK⁻or VC-TK⁻-hFlt3L (2×10⁷ pfu) or PBS were injected into the tumors on theright flank. The injections were repeated three days later. Tumors wereharvested 3 days post last injection and cell suspensions weregenerated. Cells were processed for surface labeling with anti-CD3,CD45, CD4, and CD8 antibodies. Live cells are distinguished from deadcells by using fixable dye eFluor506 (eBioscience). They were furtherpermeabilized using permeabilization kit (eBioscience), and stained forGranzyme B. For the staining of the myeloid cell population,fluorochromeconjugated antibodies against CD45.2 (104), CD11b (M1/70),Ly-6C (HK1.4), MHC II (M5/114.15.2), CD24 (M1/69), F4/80 (BM8), CD103(2E7) and CD11c (N418) were purchased from eBioscience. All antibodieswere tested with their respective isotype controls. Data were acquiredusing the LSRII Flow cytometer (BD Biosciences). Data were analyzed withFlowJo software (Treestar).

Reagents

The commercial sources for reagents were as follows: anti-mGM-CSF andanti-hFlt3L antibodies were purchased from R & D. Therapeutic anti-CTLA4(clone 9H10 and 9D9), anti-PD-L1 (clone 10F-9G2) were purchased fromBioXcell, West Lebanon, N.H. hFlt3L and mGM-CSF expression plasmids werepurchased from GE. Antibodies used for flow cytometry were purchasedfrom eBioscience (CD45.2 Alexa Fluor 700, CD3 PE-Cy7, CD4 APC-efluor780,CD8 PerCP-efluor710), Invitrogen (CD4 QDot 605, Granzyme B PE-Texas Red,Granzyme B APC). Fluorochromeconjugated antibodies against CD45.2 (104),CD11b (M1/70), Ly-6C (HK1.4), MHC II (M5/114.15.2), CD24 (M1/69), F4/80(BM8), CD103 (2E7) and CD11c (N418) were purchased from eBioscience.

Statistics

Two-tailed unpaired Student's t test was used for comparisons of twogroups in the studies. Survival data were analyzed by log-rank(Mantel-Cox) test. The p values deemed significant are indicated in thefigures as follows: *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.The numbers of animals included in the study are discussed in eachfigure legend.

Example 1 E3LΔ83N Virus (VC) Replicates in Murine and Human MelanomaCells

To test whether E3LΔ83N (the parental VC virus for the presentexperiments) or ΔE3L replicates in murine or human melanoma cells,murine B16 melanoma cells, human melanoma cells SK-MEL39, SK-MEL188, andSK-MEL90 were infected with either E3LΔ83N or ΔF3L viruses at amultiplicity of infection (MOI) of 5. Cells were collected at varioustimes post-infection (up to 50 hours post-infection). Virus yields (logPFU) were determined by titration on BSC40 cell monolayers. As shown inFIGS. 1A-1D, E3LΔ83N virus (VC) could replicate efficiently in all ofthe murine and human melanoma cells tested, whereas ΔE3L virus (VI)failed to replicate in those cell lines.

Example 2 Generation of Recombinant E3LΔ83N-TK⁻ Viruses with or withoutGM-CSF or Flt3L

It has been previously shown that oncolytic vaccinia viruses with thedeletion of thymidine kinase (TK⁻) are more attenuated and more tumorselective than TK⁺ viruses (Buller et al. 1988; Puhlmann et al., 2000).In the present disclosure, the inventors generated recombinant VCviruses comprising a TK-deletion with and without expressing human Flt3L(hFlt3L) or murine GM-CSF (mGM-CSF) under the vaccinia syntheticearly/late promoter (Pse/1) using standard recombinant virus technologythrough homologous recombination at the TK locus between the plasmid DNAand viral genomic DNA. First, the inventors constructed a plasmidcontaining specific gene of interest (SG) under the control of thevaccinia Pse/1 as well as the E. coli xanthineguanine phosphoribosyltransferase gene (gpt) under the control of vaccinia P7.5 promoterflanked by the thymidine kinase (TK) gene on either side (FIG. 2) BSC40cells were infected with VC virus at a MOI of 0.05 for 1 h, and thenwere transfected with the plasmid DNAs described above. The infectedcells were collected at 48 h. Recombinant viruses were selected throughfurther culturing in gpt selection medium including MPA, xanthine andhypoxanthine, and plaque purified (Lorenzo et al., 2004). PCR analysiswas performed to identify recombinant viruses with loss of part of theTK gene and with and without murine GM-CSF, or human Flt3L, (FIG. 3).

Oligonucleotide primers were designed to amplify DNA fragments ofdifferent sizes to identify VC recombinant viruses with differentinsertions (table in FIG. 3). For example, Primers TK-F2, which isadjacent to insertion site on the VC genome, and pCB-R3, which is on thevector, were used to check homologous insertion of target genes. PrimersTK-R4, which is missing in recombinant virus, and TK-F4, were used todistinguish the recombinant and parent viruses. Gene specific primerswere used to check the insertions of murine GM-CSF and human Flt3Lgenes. Primers TK-F5/TK-R5, which are located in the flanking region ofvaccinia TK gene (NCBI GenBank Reference NC_006998.1 (80724 . . .81257)), were used to amplify the target gene for sequence verification.Expected fragments are shown in the table in FIG. 3. Gene specificprimers mGMCSF-F1/R1 will amplify a 310 bp DNA fragment fromVC-TK⁻-mGM-CSF virus, while hFlt3L-F1/R1 will generate a 316 bp PCRfragment from VC-TK⁻-hFlt3L virus.

Primer sequence: TK-F2 (SEQ ID NO:1): TGTGAAGACGATAAATTAATGATC; TK-F4(SEQ ID NO:2): TTGTCATCATGAACGGCGGA;

TK-R4 (SEQ ID NO:3): TCCTTCGTTTGCCATACGCT;

TK-F5 (SEQ ID NO:4): GAACGGGACTATGGACGCAT;

TK-R5 (SEQ ID NO:5): TCGGTTTCCTCACCCAATCG;

pCB-R3 (SEQ ID NO:6): ACCTGATGGATAAAAAGGCG;

mGMCSF-F1 (SEQ ID NO:7): GGCATTGTGGTCTACAGCCT;

mGMCSF-R1 (SEQ ID NO:8): GTGTTTCACAGTCCGTTTCCG;

hFlt3L-F1 (SEQ ID NO:9): AACGACCTATCTCCTCCTGC;

hFlt3L-R1 (SEQ ID NO:10): GGGCTGAAAGGCACATTTGG.

Example 3 Expression of mGM-CSF from Melanoma Cells Infected withRecombinant VC-TK-mGM-CSF Virus

To test the expression of mGM-CSF from the VC-TK⁻ recombinant viruses,the inventors infected B16 murine melanoma cells and human melanomacells (SK-mel-28) with VC-TK⁻-mGM-CSF. Cell lysates and supernatantswere collected at various times (4, 8, and 24 hours) post infection.Western blot analyses were performed to determine the levels ofexpression of the transgenes. As shown in FIG. 4A, the inventorsobserved abundant levels of mGM-CSF in both the cell lysates andsupernatants.

Example 4 Expression of hFlt3L from Melanoma Cells Infected withRecombinant VC-TK-hFlt3L Virus

To test the expression of hFlt3L from the VC-TK⁻ recombinant viruses,the inventors infected B16 murine melanoma cells and human melanoma celllines with VC-TK⁻nFlt3L. Cell lysates and supernatants were collected atvarious times post infection (4, 8, and 24 hours). Western blot analysiswas performed to determine the levels of expression of the transgenes.The inventors detected abundant levels of hFlt3L in the cell lysates butnot in supernatants (FIG. 4B). This is consistent with the notion thathFlt3L is mostly associated with membranes and is not secreted.

Example 5 VC-TK, VC-TK⁻-mGM-CSF and VC-TK⁻-hFlt3L are ReplicationCompetent

The replication capacities of VC, VC-TK⁻, VC-TK⁻-mGM-CSF, andVC-TK⁻-hFlt3L in murine B16-F10 cells were determined by infecting themat a MOI of 0.01. Cells were collected at various times post-infection(24, 48, and 72 hours) and viral yields (log pfu) were determined bytitration on BSC40 cells. VC replicated efficiently in B16-F10 cellswith viral titers increasing by 20,000-fold at 72h post-infection.(FIGS. 5A and 5B). Deletion of TK gene resulted in the 3-fold decreasein viral replication in B16 melanoma cells compared with VC. Inaddition, E3LΔ83N-TK⁻-mGM-CSF and E3LΔ83N-TK⁻-hFlt3L were alsoreplication competent in murine B16 cells, with an increase of viraltiters by 2800-fold and 1000-fold at 72 h post infection, respectively(FIGS. 6A and 6B). Thus, in this Example, the inventors have shown thatVC-TK⁻, VC-TK⁻-mGM-CSF and VC-TK⁻-hFlt3L are all replication competentin tumor cells.

Example 6 Intratumoral Injection of VC-TK, VC-TK⁻-mGM-CSF, orVC-TK⁻-hFlt3L Leads to Eradication of Injected Tumors and Delayed TumorGrowth at the Contralateral Non-Injected Tumors

To test the in vivo tumor killing activities of the recombinant virusesand vector control, murine B16 melanoma cells were implanted to C57B/6mice, with 1×10⁵ cells to the left flank and 5×10⁵ cells to the rightflank. The inventors performed intratumoral injection of VC-TK⁻,VC-TK⁻-mGM-CSF, or VC-TK⁻-hFlt3L (2×10⁷ pfu) twice weekly to the largertumor (about 3-4 mm in diameter) on the right flank. PBS mock-treatmentcontrol was included in the study. Bilateral tumor sizes were measuredtwice a week and mice were monitored for survival. When the tumor sizesreached 1 cm in diameter, the mice were euthanized. The experimentalscheme is shown in FIG. 6. The inventors observed that the PBSmock-treated tumors grew very quickly and mice died with a mediumsurvival of 15 days (FIG. 7A, B). 10/10 of the E3LΔ83N-TK⁻-injectedtumors regressed (FIG. 7C).

However, the contralateral tumors continued to grow (FIG. 7D) and all ofthe mice died with a median survival of 18 days (P<0.05, compared withPBS-treated group) (FIG. 7M). The addition of mGM-CSF to the VC-TK⁻vector did not result in prolonged survival compared with VC-TK⁻ vector(FIG. 7M). However, intratumoral injection of VC-TK⁻-hFlt3L not onlyeradicated 8/10 injected tumors but also resulted in delayed tumorgrowth in the contralateral tumors and extended medium survival to 22days (P<0.01, compared with PBS-treated group; P=0.02, compared withVC-TK⁻-mGM-CSF-treated group) (FIG. 7E, F, M). These results demonstratethat although intratumor injection of attenuated replication competentVC-TK⁻, VC-TK⁻-mGM-CSF, or VC-TK⁻-hFlt3L can effectively eradicateinjected tumors, intratumoral delivery of VC-TK⁻-hFlt3L is moreefficacious than VC-TK⁻-mGM-CSF in delaying the growth of contralateraltumor and extending survival.

Example 7 The Combination of Intratumoral Delivery of RecombinantVC-TK⁻-mGM-CSF, or VC-TK⁻-hFlt3L with Systemic Delivery of ImmuneCheckpoint Blocking Agent Leads to More Efficient Tumor Eradication andLonger Survival than Either Treatment Alone

It has been shown that systemic delivery of anti-CTLA-4 antibody isincapable of controlling B16 melanoma growth. To test whetherintratumoral delivery of oncolytic viruses would overcome the resistanceto immune checkpoint blocking agents, the inventors used murine B16bilateral tumor implantation model in which the larger tumors on theright flank of the mice were injected twice weekly with eitherVC-TK⁻-mGM-CSF, or VC-TK⁻-hFlt3L with or without intraperitonealdelivery of anti-CTLA-4 antibody. Tumor sizes were measured twice weeklyand the survival of mice was monitored. The inventors found that thecombination of intratumoral delivery of either VC-TK⁻-mGM-CSF, orVC-TK-hFlt3L with systemic delivery of anti-CTLA-4 antibody lead to theeradication of 10/10 injected tumors, and significant delay of thegrowth of contralateral non-injected tumors, as well as completeeradication of tumors in 40-60% of the cases (FIG. 7I-M). Similarly tothe results observed in Example 6, intratumoral delivery ofVC-TK⁻-hFlt3L in combination with anti-CTLA-4 antibody was moreefficacious than VC-TK⁻-mGM-CSF in combination with anti-CTLA-4 antibodyin delaying the growth of contralateral tumor and extending survival oftreated mice. Taken together, these results indicate that intratumoraldelivery of attenuated replication competent oncolytic viruses caninduce antitumor immunity, which is amplified in the presence ofanti-CTLA-4 antibody.

Example 8 Intratumoral Delivery of Heat-Inactivated VC-TK⁻-mGM-CSF isMore Efficacious in Eradiating Tumors and Generating AntitumoralAdaptive Immunity than Live VC-TK⁻-mGM-CSF

The inventors previously reported that Heat-inactivated MVA is moreefficacious than MVA in eradicating injected tumors and inhibiting ordelaying the growth of non-injected distant tumors in a bilateralB16-F10 bilateral tumor implantation model (See InternationalApplication PCT/US2016/19663 filed by the inventors and co-workers onFeb. 25, 2016; and provisional application No. 62/149,484 filed on Apr.17, 2015 and its corresponding international application,PCT/US2016/028184 filed Apr. 18, 2016. These applications are hereinincorporated by reference in their entirety). Because most of oncolyticviruses in clinical trials, including T-VEC, which has been approved forthe treatment of metastatic melanoma, are replication competent, theinventors performed a head-to-head comparison between VC-TK⁻-mGM-CSF andHeat-inactivated VC-TK⁻-mGM-CSF in a bilateral B16-F10 implantationmodel. VC-TK⁻-mGM-CSF is similar to JX594 in that it has TK deletion andGM-CSF transgene. Although VC-TK-mGM-CSF replicates efficiently in B16melanoma cells, it is more attenuated than WT vaccinia in animals andpossibly in humans due to the deletion of the Z-DNA-binding domain of E3(Brandt and Jacobs, JVI, 2001). Therefore, it is anticipated thatVC-TK-mGM-CSF will be safer than JX594 for human use. The inventorshypothesized that, similar to Heat-MVA, Heat-VC-TK⁻-mGM-CSF would be astronger activator of antitumor immunity than live VC-TK⁻-mGM-CSF due toits ability to induce type I IFN in DCs and cancer cells. To test that,the inventors performed the following experiment. Briefly, B16-F10melanoma cells were implanted intradermally to the left and right flanksof C57B/6 mice (5×10⁵ to the right flank and 1×10⁵ to the left flank). 7days after tumor implantation, 2×10⁷ pfu of live VC-TK⁻-mGM-CSF or anequivalent amount of Heat-VC-TK⁻-mGM-CSF was intratumorally injected tothe larger tumors on the right flank. The tumor sizes were measured andthe tumors were injected twice a week. Mouse survival was monitored. Itwas found that in mice treated with PBS, tumors grow rapidly at theright flank, which resulted in early death (FIG. 8 A, B, O).Intratumoral injection of either Heat-VC-TK⁻-mGM-CSF or liveVC-TK⁻-mGM-CSF resulted in delaying of tumor growth and improvedsurvival compared with PBS (FIG. 8O, ***, P<0.001 for VC-TK⁻-mGM-CSF vs.PBS, ****, P<0.0001 for Heat-VC-TK⁻-mGM-CSF vs. PBS). Intratumoralinjection of Heat-VC-TK⁻-mGM-CSF is more efficacious than VC-TK⁻-mGM-CSFin eradicating injected tumors (8/9 tumor free for Heat-VC-TK⁻-mGM-CSFvs. 4/9 tumor free for VC-TK⁻-mGM-CSF) and delaying or inhibiting thegrowth of non-injected tumors at the contralateral side (7/9 tumor freefor Heat-VC-TK⁻-mGM-CSF vs. 5/9 tumor free for VC-TK⁻-mGM-CSF) (FIG.8C-F). The inventors observed improved survival inHeat-VC-TK⁻-mGM-CSF-treated mice compared with VC-TK⁻-mGM-CSF-treatedmice (FIG. 8O, *, P=0.014). These results indicate that viralreplication is not necessary for achieving antitumor effects. While, inthis specific example, the inventors used heat inactivation toinactivate the virus, inactivation can be done by other methods. Forexample, another method of virus inactivation comprises use ofultraviolet irradiation.

Intratumoral injection of Heat-VC-TK⁻-mGM-CSF leads to antitumorimmunity possibly through the induction of STING-mediated type I IFNresponses, activation of Batf3-dependent dendritic cells (DC) andrecruitment and activation of anti-tumor CD8⁺ and CD4⁺ T cells, as wellas increase of ratios of CD8⁺/Treg and CD4⁺ effector/Treg as theinventors of the present disclosure have demonstrated for Heat-MVA.

The co-administration of live and Heat-VC-TK⁻-mGM-CSF into tumors didnot result in improved antitumor responses compared toHeat-VC-TK⁻-mGM-CSF alone (FIG. 8G, H, O). The inventors reasoned thatthe live virus might block the host's immune responses and thereforeactivities of the Heat-VC-TK⁻-mGM-CSF, which may mitigate the beneficialeffects of GM-CSF. Studies are ongoing to evaluate whether theco-administration of live and Heat-VC-TK⁻-hFlt3L might be moreefficacious than Heat-VC-TK⁻-hFlt3L alone. Studies are also ongoing tofurther attenuate replication competent VC-TK⁻-hFlt3L through deletionof candidate genes that interfere with the cytosolic DNA-sensingpathway.

Example 9 The Combination of Intratumoral Injection of VC-TK⁻-mGM-CSF orHeat-Inactivated VC-TK⁻-mGM-CSF with Intraperitoneal Delivery of ImmuneCheckpoint Blocking Agent Leads to Synergistic Therapeutic Effects

The inventors next investigated whether intratumoral injection of liveor Heat-inactivated VC-TK⁻-mGM-CSF enhances therapeutic effects ofanti-PD-L1 antibody in a bilateral B16-F10 melanoma model, whichsimulates an individual with metastatic disease. Briefly, B16-F10melanoma cells were implanted intradermally to the left and right flanksof C57B/6 mice (5×10⁵ to the right flank and 1×10⁵ to the left flank). 8days after tumor implantation, the inventors intratumorally injectedVC-TK⁻-mGM-CSF (2×10⁷ pfu), or an equivalent amount of Heat-inactivatedVC-TK⁻-mGM-CSF, or the combination of live (1×10⁷ pfu) andHeat-VC-TK⁻-mGM-CSF (an equivalent of 1×10⁷ pfu) to the larger tumors onthe right flank twice weekly, with intraperitoneal delivery of eitherisotype control, or with anti-PD-L1 antibody (200 μg per mouse) twiceweekly.

The combination of intratumoral injection of live VC-TK⁻-mGM-CSF andsystemic delivery of anti-PD-L1 antibody resulted in a significantimprovement of mouse survival (FIG. 8O, *, P=0.02 forVC-TK⁻-mGM-CSF+anti-PD-L1 vs. VC-TK⁻-mGM-CSF). 67% of the mice (6/9)treated with live VC-TK⁻-mGM-CSF+anti-PD-L1 antibody were tumor free,whereas only 22% of the mice (2/9) treated with live VC-TK⁻-mGM-CSF weretumor free at the end of the experiment (FIG. 8O). All of the mice (9/9)treated with Heat-VC-TK⁻-mGM-CSF+anti-PD-L1, 89% of mice (8/9) treatedlive+Heat-VC-TK⁻-mGM-CSF+anti-PD-L1 are alive at the end of experiment(day 67 post virus injection) (FIG. 8O). These results furtherdemonstrated that, in the combination therapy setting,Heat-VC-TK⁻-mGM-CSF or live VC-TK⁻-mGM-CSF-induced antitumor immunitycan be further amplified in the presence of anti-PD-L1 antibody.

Example 10 The Surviving Mice Treated with Heat-InactivatedVC-TK-mGM-CSF with or without Anti-PD-L1, or Treated with LiveVC-VC-TK-mGM-CSF with Anti-PD-L1 have Developed Cross-ProtectiveImmunity Against a Heterologous Tumor

The inventors next tested whether the surviving mice that aresuccessfully treated with viruses with or without anti-PD-L1 antibodyfor initial B16-F10 tumor have development cross-protective immunityagainst a heterologous tumor, in this case, MC38 colon carcinoma cells.This experiment included the following groups of mice: (i) survivingmice treated with live VC-TK⁻-mGM-CSF (n=2), (ii) surviving mice treatedwith live VC-TK⁻-mGM-CSF+anti-PD-L1 (n=6), (iii) surviving mice treatedwith Heat-VC-TK⁻-mGM-CSF (n=7), (iv) surviving mice treated withHeat-VC-TK⁻-mGM-CSF+anti-PD-L1 (n=9), (v) surviving mice treated withlive+Heat-VC-TK⁻-mGM-CSF+anti-PD-L1 (n=6), (vi) surviving mice treatedwith live+Heat-VC-TK⁻-mGM-CSF+anti-PD-L1 (n=8), and (vii) nave mice thathave never been exposed either to tumors or viruses (n=5). All of themice were challenged with intradermal implantation of a lethal dose ofMC38 (1×10⁵ cells) and the tumor sizes were measured twice weekly andthe survival of the mice were monitored daily. The inventors observedthat although all of the nave mice developed MC38 and die at expectedtime with a median survival of 30 days, 2/2 of the surviving micetreated with live VC-TK⁻-mGM-CSF died at a later time with a mediansurvival of 42 days (FIG. 9, p<0.05; VC-TK⁻-mGM-CSF vs. nave mice).Surprisingly, the majority of the rest of surviving mice rejected MC38challenge at 100 days post tumor implantation (FIG. 9). These resultsindicate that the surviving mice treated with Heat-VC-TK⁻-mGM-CSF withor without anti-PD-L1 antibody have developed systemic immunity againsta heterologous tumor. Such immunity is weaker in mice previously treatedwith live VC-TK⁻-mGM-CSF, although only two mice were in this group.Future studies will expand the numbers of mice successfully treated withlive VC-TK⁻-mGM-CSF vs. Heat-VC-TK⁻-mGM-CSF in an unilateral B16-F10tumor implantation model and then assess cross-protective immunityagainst MC38, or another heterologous tumor such as MB49 bladder cancer.

Example 11 Intratumoral Injection of VC-TK⁻-hFlt3L Virus is MoreEffective than VC-TK⁻ Virus in the Proliferation and Activation of CD8⁺and CD4⁺ T Cells in the Non-Injected Tumors

To assess whether intratumoral injection of VC-TK⁻ or VC-TK⁻-hFlt3L inB16-F10 melanomas leads to activation and proliferation of CD8⁺ and CD4⁺T cells, 2.5×10⁵ B16-F10 melanoma cells were intradermally implanted tothe left flank and 5×10⁵ B16-F10 melanoma cells to the right flank of6-8 weeks old C57B/6. 7 days post-implantation, VC-TK⁻ or VC-TK⁻-hFlt3L(2×10⁷ pfu) or PBS was injected into the larger tumors on the rightflank. The injection was repeated three days later. Both the injectedand non-injected tumors were harvested on day 7 after first injection,and cell suspensions were generated. The live immune cell infiltrates inthe injected and non-injected tumors were analyzed by FACS. There was adramatic increase in CD8⁺ T cells expressing Granzyme B in the injectedtumors, from 51% in PBS-treated tumors to 81% in VC-TK⁻-treated tumorsand 83% in VC-TK⁻-hFlt3L-treated tumors (FIG. 10A, 10C, p<0.001; VC-TK⁻or VC-TK⁻-hFlt3L vs. PBS). In the non-injected tumors, there was also asincrease in CD8⁺ T cells expressing Granzyme B from 48% in PBS-treatedmice to 54% in VC-TK⁻ treated and 70% in VC-TK⁻-hFlt3L-treated mice(FIG. 10A, 10C, p<0.05; VC-TK⁻-hFlt3L vs. PBS). These results indicatethat intratumoral injection of either VC-TK⁻ or VC-TK⁻-hFlt3L led toincreased activated CD8⁺ T cells in the injected tumors, andintratumoral injection of VC-TK⁻-hFlt3L but not VC-TK⁻ led tosignificantly increased activated CD8⁺ T cells in the non-injectedtumors.

Similar changes were observed for CD4⁺ T cells in the injected andnon-injected tumors from mice treated with either VC-TK⁻ orVC-TK⁻-hFlt3L compared with those treated with PBS. Granzyme B⁺CD4⁺ Tcells rose from 9% in PBS-treated tumors to 74% in VC-TK⁻-treated tumorsand 71% in VC-TK⁻-hFlt3L-treated tumors (FIG. 10B, 10D, p<0.001; VC-TK⁻or VC-TK⁻-hFlt3L vs. PBS). In the non-injected tumors, there was also asincrease in CD4⁺ T cells expressing Granzyme B from 11% in PBS-treatedmice to 13% in VC-TK⁻-treated and 32% in VC-TK⁻-hFlt3L-treated mice(FIG. 10B, 10D, p<0.01; VC-TK⁻-hFlt3L vs. PBS). These results indicatethat intratumoral injection of either VC-TK⁻ or VC-TK⁻-hFlt3L led toincreased activated CD4⁺ T cells in the injected tumors, andintratumoral injection of VC-TK⁻-hFlt3L but not VC-TK⁻ led to increasedactivated CD4⁺ T cells in the non-injected tumors.

Example 12 Intratumoral Injection of VC-TK⁻-hFlt3L Results in theIncrease of CD103⁺ Dendritic Cells in the Non-Injected Tumors

The inventors next analyzed dendritic cell (DC) populations in bothinjected and non-injected tumors. Tumor infiltrating DCs arecharacterized as CD45⁺Ly6C-MHC-II+CD24^(hi)F4/80^(lo) cells (Broz etal., Cancer Cell, 2014). Among the CD24^(hi) DCs, there are two DCpopulations, CD11b⁺ DC (also known as DC1) and CD103⁺ DC (also known asDC2). FIG. 11 shows the gating strategy for these DC populations. CD45⁺live cells were further separated based on the expression of MHC-II. TheMHC-II^(hi) cells were stained for DC marker CD24 and tumor-associatedmacrophage marker F4/80. CD24^(hi)F4/80^(lo) cells were furtherseparated into CD103⁺ DCs and CD11b⁺ DCs based on their expressions ofCD103 and CD11b.

CD103+ DCs is a subset of peripheral DCs that are specialized incross-presenting antigens. Batf3 is a transcription factor that isimportant for the differentiation of CD103+ DCs. CD103+ DCs playimportant roles in host anti-tumor immunity. The inventors of thepresent disclosure have previously shown that Batf3-dependent CD103⁺ DCsare required for inactivated MVA-mediated antitumor effects(WO2016/168862). Here, the inventors investigated the percentages ofCD103⁺ DCs out of CD45+ cells in both injected and non-injected tumors.

B16-F10 melanoma cells (2.5×10⁵) were intradermally implanted to theleft flank and 5×10⁵ B16-F10 melanoma cells to the right flank of 6-8weeks old C57B/6. 7 days post-implantation, Heat-MVA, VC-TK⁻,VC-TK⁻-mGM-CSF, or VC-TK⁻-hFlt3L (2×10⁷ pfu) or PBS was injected intothe larger tumors on the right flank. The injection was repeated threedays later. Both the injected and non-injected tumors were harvested onday 7 after first injection, and cell suspensions were generated. Thelive myeloid cell infiltrates in the injected and non-injected tumorswere analyzed by FACS. The inventors observed that intratumoralinjection of Heat-MVA, or VC-TK⁻, or VC-TK⁻-mGM-CSF, or VC-TK⁻-hFlt3Lresulted in the reduction of percentages of CD103⁺ DCs out of CD45⁺cells from 0.2% in PBS-mock treated tumors to 0.03%, 0.03%, 0.05%, and0.12% in Heat-MVA, VC-TK⁻, VC-TK⁻-mGM-CSF, or VC-TK⁻-hFlt3L-treatedtumors (FIG. 12A; P<0.05, Heat-MVA, or VC-TK⁻, or VC-TK⁻-mGM-CSF vs.PBS). In the non-injected tumors, intratumoral injection of Heat-MVA, orVC-TK⁻, or VC-TK⁻-mGM-CSF, or VC-TK⁻-hFlt3L resulted in the increase ofpercentages of CD103⁺ DCs out of CD45⁺ cells from 0.15% in PBS-mocktreated mice to 0.32%, 0.26%, 0.21%, and 0.39% in Heat-MVA, VC-TK⁻,VC-TK⁻-mGM-CSF, or VC-TK⁻-hFlt3L-treated mice (FIG. 12A; P<0.05,VC-TK⁻-hFlt3L vs. PBS). These results indicate that CD103⁺ DCs undergodynamic changes after intratumoral injection with viruses includingdecrease of percentages of CD103⁺ DCs out of CD45+ cells in the injectedtumors, and intratumoral injection of VC-TK⁻-hFlt3L leads to thesignificant increase of percentages of CD103⁺ DCs out of CD45+ cells inthe contralateral non-injected tumors. These results are consistent withthe understanding that hFlt3L is an important growth factor for thedifferentiation and proliferation of CD103⁺ DCs.

The percentages of CD11b⁺ DCs out of CD45⁺ cells in both injected andnon-injected tumors were also investigated. It was found thatintratumoral injection of VC-TK⁻ led to the increase of the percentagesof CD11b⁺ DCs out of CD45⁺ cells from 0.5% in PBS-treated tumors to 0.8%in VC-TK−-treated tumors (FIG. 12B, P<0.05, VC-TK⁻ vs. PBS).Intratumoral injection of viruses does not seem to affect CD11b⁺ DCpopulations in the non-injected tumors (FIG. 12B). Taken together, theseresults indicate that intratumoral injection of VC-TK⁻-hFlt3L leads tothe significant increase of percentages of CD103⁺ DCs out of CD45⁺ cellswithout affecting the percentages of CD11b⁺ DCs out of CD45⁺ cells inthe contralateral non-injected tumors.

Example 13 Intratumoral Injection of VC-TK⁻ is Effective in a BilateralTriple-Negative Breast Cancer 4T1 Tumor Implantation Model

In addition to B16-F10 murine melanoma model, the inventors investigatedwhether intratumoral injection of oncolytic virus VC-TK− has efficacy inthe treatment of triple-negative breast cancer (TNBC) 4T1 bilateraltumor implantation model. Briefly, 4T1 murine triple negative breastcancer (TNBC) cells were implanted intradermally to the left and rightflanks of BALB/c mice (2.5×10⁵ to the right flank and 5×10⁴ to the leftflank). 5 days post tumor implantation, the larger tumors on the rightflank were injected with either VC-TK⁻ virus (2×10⁵ pfu) or with anequivalent amount of Heat-inactivated MVA twice weekly. Mice weremonitored daily and tumor sizes were measured twice a week. The survivalof mice was monitored. The initial tumor volumes of the injected andnon-injected tumors were shown (FIGS. 13 A and B). The tumor volumes ofthe injected and non-injected tumors at 18-day post treatment were shown(FIGS. 13 C and D). It was found that intratumoral injection of VC-TK⁻led to dramatic decrease of tumor volumes of the injected tumorscompared with PBS-treated tumors (FIG. 13C; P<0.0001, VC-TK⁻ vs. PBS)and also decrease of non-injected tumors volumes compared withPBS-treated mice (FIG. 13D; P<0.05, VC-TK⁻ vs. PBS). More importantly,the mean survival of mice was extended from 18 days in PBS-treated miceto 21 days in VC-TK⁻-treated mice (FIG. 13E; P=0.0001, VC-TK⁻ vs. PBS).The anti-tumor effect of VC-TK⁻ is similar to Heat-iMVA in thisbilateral 4T1 tumor implantation model (FIG. 13, A-E). That is differentfrom what we observed in B16-F10 bilateral tumor implantation model(FIGS. 8, C-F and O), in which VC-TK⁻-mGM-CSF is less effective thanHeat-inactivated VC-TK⁻-mGM-CSF. These might be related to thedifferences in tumor subtypes and the populations of immune cells in thetumor microenvironment. Future studies will compare the efficacies ofreplication competent oncolytic virus with inactivated virus in othertumor models including prostate cancer and bladder cancer models.Because VC-TK⁻-hFlt3L is more effective than VC-TK⁻-mGM-CSF shown inExample 6 (FIG. 8 E-H, M), it is expected that intratumoral injection ofoncolytic VC-TK⁻-hFlt3L would also be effective in treating 4T1 murinebreast cancer.

Example 14 Intratumoral Injection of VC-TK⁻-mGM-CSF is Effective in aLarge Established B16-F10 Unilateral Tumor Implantation Model

The inventors compared the anti-tumor efficacy of intratumoral injectionof replication competent VC-TK⁻-mGM-CSF with Heat-inactivated MVA(Heat-iMVA) in a large established B16-F10 unilateral tumor implantationmodel. In this experiment, B16-F10 melanoma (5×10⁵ cells in a volume of50 μl) were implanted intradermally into the shaved skin on the rightflank of WT C57BL/6J mice. After 9 days post implantation, tumor sizeswere measured and tumors that are 5-6 mm in diameter were injected withHeat-iMVA (equivalent of 2×10⁷ pfu of MVA in a volume of 50 μl) or withVC-TK⁻-mGM-CSF (2×10⁷ pfu), or with PBS twice weekly. Mice weremonitored daily and tumor sizes were measured twice a week. Intratumoralinjection of VC-TK⁻-mGM-CSF was efficacious in delaying tumor growth andeven eradicating tumors in a small percentage of treated mice. It alsoextended the median survival from 6 days in PBS-treated mice to 17 daysin VC-TK⁻-mGM-CSF-treated mice (FIG. 14, A, C-E, P<0.0001,VC-TK⁻-mGM-CSF vs. PBS). Intratumoral injection of Heat-iMVA in largeestablished tumors were also effective and the median survival ofHeat-iMVA-treated mice was extended to 27 days (FIGS. 14, B, D, and E).These results indicate that intratumoral injection of oncolyticVC-TK⁻-mGM-CSF is effective in treating large established B16-F10 in aunilateral implantation model. Because VC-TK⁻-hFlt3L is more effectivethan VC-TK⁻-mGM-CSF shown in Example 6 (FIG. 8 E-H, M), it is expectedthat intratumoral injection of oncolytic VC-TK⁻-hFlt3L would also beeffective in treating large established B16-F10 in a unilateralimplantation model.

All patent and literature documents cited herein are incorporated byreference in their entirety for all purposes.

What is claimed is:
 1. A method for eliciting an immune response in asubject in need thereof, the method comprising delivering to the subjecta therapeutically effective amount of a composition comprising arecombinant vaccinia virus selected from the group consisting of: (i)E3LΔ83N-TK⁻-hFlt3L; (ii) E3LΔ83N-TK⁻; (iii) E3LΔ83N-TK⁻-GM-CSF; and (iv)combinations thereof, wherein the virus is in replicative or inactivatedform.
 2. The method of claim 1, wherein the recombinant vaccinia viruscomprises E3LΔ83N-TK⁻-hFlt3L in replicative or inactivated form.
 3. Themethod of claim 1, wherein eliciting the immune response in the subjectcomprises one or more of the following immunological effects selectedfrom: increased activation of CD8⁺ T cells; increased activation of CD4⁺T cells; and increased CD103⁺ dendritic cells.
 4. The method of claim 1,wherein the recombinant vaccinia virus is delivered to the subjectparenterally, intravenously, intratumorally, intraperitoneally,intrathecally, or intracranially.
 5. The method of claim 1, wherein therecombinant vaccinia virus is heat-inactivated.
 6. A method for treatinga malignant solid tumor in a subject in need thereof, the methodcomprising delivering to the cells of the tumor a recombinant vacciniavirus in replicative or inactivated form selected from the groupconsisting of: (i) E3LΔ83N-TK⁻-hFlt3L; (ii) E3LΔ83N-TK⁻; (iii)E3LΔ83N-TK⁻-GM-CSF; and (iv) combinations thereof, in an amounteffective to induce the immune system of the subject to mount an immuneresponse against the tumor.
 7. The method of claim 6, wherein therecombinant vaccinia virus comprises E3LΔ83N-TK⁻-hFlt3L in replicativeor inactivated form.
 8. The method of claim 6 wherein the tumor isprimary or metastatic melanoma, breast carcinoma, or colon carcinoma. 9.The method of claim 6, wherein the immune response is systemic.
 10. Themethod of claim 6, wherein the immune response is effective toaccomplish one or more of the following: reduction of the size of thetumor; eradication of the tumor; inhibition of growth of the tumor;inhibition of metastasis of the tumor; or reduction or eradication ofmetastatic tumor.
 11. The method of claim 6, wherein the tumor includestumor located at the site of delivery, or tumor located at the site ofdelivery and elsewhere in the body of the subject.
 12. The method ofclaim 6, wherein the immune response comprises one or more of thefollowing: increase in cytotoxic CD8⁺ T cells within the tumor and/or intumor-draining lymph nodes; induction of maturation of dendritic cellsinfiltrating the tumor through induction of type I IFN; induction ofactivated CD4⁺ effector T cells in the subject recognizing tumor cellswithin the tumor or systemically; and increase of CD103⁺ dendritic cellsin non-injected tumors of the subject.
 13. The method of claim 6,further comprising conjointly administering an immune checkpointblocking agent in an amount effective to block immune suppressivemechanisms within the tumor elicited by tumor cells, stromal cells, ortumor infiltrating immune cells.
 14. The method of claim 13, wherein theimmune checkpoint blocking agent comprises CTLA-4 inhibitors, CD80inhibitors, CD86 inhibitors, PD-1 inhibitors, PD-L1 inhibitors, PD-L2inhibitors, LAG3 inhibitors, B7-H3 inhibitors, B7-H4 inhibitors, TIM3inhibitors, ICOS inhibitors, II DLBCL inhibitors, BTLA inhibitors,ipilimumab, nivolumab, pembrolizumab, pidilizumab, AMP-224, MPDL3280A,BMS-936559, MEDI4736, MSB 00107180, or any combination thereof.
 15. Themethod of claim 6, wherein the subject has been previously treated ordosed with an amount of an immune checkpoint blocking agent effective toblock immune suppressive mechanisms within the tumor elicited by tumorcells, stromal cells, or tumor infiltrating immune cells.
 16. The methodof claim 15, wherein the immune checkpoint blocking agent comprisesCTLA-4 inhibitors, CD80 inhibitors, CD86 inhibitors, PD-1 inhibitors,PD-L1 inhibitors, PD-L2 inhibitors, LAG3 inhibitors, B7-H3 inhibitors,B7-H4 inhibitors, TIM3 inhibitors, ICOS inhibitors, II DLBCL inhibitors,BTLA inhibitors, ipilimumab, nivolumab, pembrolizumab, pidilizumab,AMP-224, MPDL3280A, BMS-936559, MEDI4736, MSB 00107180, or anycombination thereof.
 17. The method of claim 6, wherein the recombinantvaccinia virus is delivered to the subject parenterally, intravenously,intratumorally, intraperitoneally, intrathecally, or intracranially. 18.The method of claim 6, further comprising combining the treatment withan additional treatment modality comprising surgery, chemotherapy,targeted therapy, and/or radiation.
 19. The method of claim 6, whereinthe recombinant vaccinia virus is heat-inactivated.